WO1999058725A1 - Process for obtaining amplified fragments of nucleic acid of viral and sub-viral particles which contain rna - Google Patents

Process for obtaining amplified fragments of nucleic acid of viral and sub-viral particles which contain rna Download PDF

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Publication number
WO1999058725A1
WO1999058725A1 PCT/ES1998/000128 ES9800128W WO9958725A1 WO 1999058725 A1 WO1999058725 A1 WO 1999058725A1 ES 9800128 W ES9800128 W ES 9800128W WO 9958725 A1 WO9958725 A1 WO 9958725A1
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Prior art keywords
viral
pcr
reverse transcription
temperature
reaction
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PCT/ES1998/000128
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Spanish (es)
French (fr)
Inventor
Fernando Ponz Ascaso
Vicente Torres Pascual
Begoña BLANCO URGOITI
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Instituto Nacional De Investigacion Y Tecnologia Agraria Y Alimentaria (Inia)
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Priority to ES09602551A priority Critical patent/ES2121698B1/en
Application filed by Instituto Nacional De Investigacion Y Tecnologia Agraria Y Alimentaria (Inia) filed Critical Instituto Nacional De Investigacion Y Tecnologia Agraria Y Alimentaria (Inia)
Priority to AU70446/98A priority patent/AU7044698A/en
Priority to PCT/ES1998/000128 priority patent/WO1999058725A1/en
Publication of WO1999058725A1 publication Critical patent/WO1999058725A1/en

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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844Nucleic acid amplification reactions
    • C12Q1/686Polymerase chain reaction [PCR]
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6804Nucleic acid analysis using immunogens

Definitions

  • This invention relates to a method for obtaining amplified fragments of virus nucleic acid with RNA genome and sub-viral particles containing RNA.
  • the resulting amplified fragments can be subsequently analyzed to detect, identify or molecularly typify said viruses and subviral particles.
  • PCR polymerase chain reaction
  • 2 such methods comprise obtaining the amplified nucleic acid fragment and subsequent analysis thereof.
  • the use of spectrophotometric and / or electrophoretic techniques for the analysis of said fragment leads to the detection and / or identification of the pathogen, while the structural analysis of the fragment amplified either by sequencing techniques or by polymorphism analysis techniques of Length of restriction fragments (RFLP) or single chain conformation (SSCP), allows the molecular typing of the pathogen and the conduct of molecular epidemiology studies.
  • RFLP polymorphism analysis techniques of Length of restriction fragments
  • SSCP single chain conformation
  • Some methods for obtaining amplified DNA fragments combine the capture of the pathogen with the enzymatic amplification by PCR.
  • PCR amplification is preceded by a reverse transcription step in order to convert RNA to DNA (substrate for PCR).
  • Methods include performing a step of lysis of the captured viral particle and release of the viral nucleic acid before performing the enzyme amplification or, where appropriate, reverse transcription (e.g., US Patent 5,077). .192), while other methods dispense with said stage of lysis and prior release of nucleic acid (Spanish patents ES 9201232 and ES 9302657).
  • obtaining an amplified nucleic acid fragment comprises: (a) the immunocapture of the pathogen in the wells of a microtiter plate,
  • the present invention provides a solution to the previously raised problems consisting of a process for obtaining amplified fragments of nucleic acid from viral or subviral particles containing RNA, which combines (a) the capture of the pathogen with (b) the addition, in a single operation, of all the reagents involved in the reverse transcription reaction and in the enzymatic amplification reaction by PCR, and with (c) the development of appropriate thermal cycles to perform, in the first place, the transcription reaction Inverse, and secondly, without the need to uncover the container containing the sample to be tested and without the need to add any additional reagents, but simply by modifying the temperature, carry out the PCR enzymatic amplification reaction of the nucleic acid fragment.
  • the amplified fragment can be subjected to spectrophotometric, electrophoretic techniques, or to structural analysis to detect, quantify, identify or molecularly typify the viral or subviral particle in question.
  • This method allows the use of a larger volume of the solution containing the initial viral RNA to perform reverse transcription (typically 80 ⁇ l), that is, A larger part of the sample and the yield and sensitivity increase, the risk of accidental contamination is reduced, the number of sample handling operations is reduced, automation is facilitated and the number of samples to be handled can be increased.
  • Figure 1 shows a photograph of an electrophoresis gel showing the presence of an 879 nucleotide fragment of the potato Y virus genome (PVY), corresponding to the amplified fragment according to the procedure provided by this invention and described in the Example 1.
  • Figure 2 shows a photograph of an electrophoresis gel showing the presence of an 879 nucleotide fragment of the PVY genome, corresponding to the amplified fragment according to the procedure described in Example 1, compared to the same fragment obtained following the procedure described in Spanish patent ES 9201232.
  • the method for obtaining amplified nucleic acid fragments of viral or subviral particles containing RNA present in a sample suspected of containing such particles, object of this invention comprises the steps of: a) adding said sample to the tubes of a thermal cycler and incubating the assembly under suitable conditions to capture said viral or subviral particles in the thermocycler tubes; b) remove the supernatant from the tubes and wash the set of viral or sub-viral particles captured in the tubes of the thermal cycler; c) adding to said tubes, which contain the captured particles, all the reagents necessary to carry out the reverse transcription reaction and the enzymatic amplification reaction by PCR; d) insert the tubes into the thermal cycler; and e) program a suitable thermal cycle in the thermal cycler so that the reverse transcription reaction of the RNA takes place in each tube first, and subsequently the enzymatic PCR amplification reaction of the nucleic acid fragment after thermal deactivation of the reverse transcriptase, for which said thermal cycle comprises: heating the thermocycler tubes at
  • A) Particle capture The first stage of the procedure involves the capture of the viral or subviral particle that contains RNA in the tubes of a thermal cycler.
  • viral particle in the sense used in this description, includes any genome virus.
  • RNA that replicates inside protoplasts, eukaryotic or prokaryotic cells, and at the expense of some enzyme of said protoplasts or cells for example, viruses 7 animals, plant viruses, bacterial viruses and fungal viruses.
  • a particular case of such viral or sub-viral particles is viral or sub-viral pathogens, a term in which viruses with an RNA genome are included and sub-viral particles that comprise RNA that can cause any pathology in animals, including man, or in plants and mushrooms.
  • the tube where the viral or sub-viral particles can be captured is a conventional tube, of those used in the thermal cyclers commonly used to carry out the enzymatic amplification of DNA fragments by PCR.
  • viral or subviral particles will be found in a sample that may be environmental, for example, a water or air sample, or a biological sample, the term of which includes eukaryotic, prokaryotic, protoplasts, mycelium, animal tissues and organs and vegetables, in vitro culture media of any of the above entities, plant or animal systems, plant or animal organisms, as well as any excretion, secretion or transformation product of any of the above entities.
  • a sample may be environmental, for example, a water or air sample, or a biological sample, the term of which includes eukaryotic, prokaryotic, protoplasts, mycelium, animal tissues and organs and vegetables, in vitro culture media of any of the above entities, plant or animal systems, plant or animal organisms, as well as any excretion, secretion or transformation product of any of the above entities.
  • the sample capable of containing them which has previously been homogenized, where appropriate, for example, in a suitable buffer, is added to the tube of the thermal cycler, incubating the assembly at the temperature and during adequate time allowing the capture of said particles.
  • the tube-sample set is incubated at temperatures between 0 and 50 ° C, for a period of time 8 normally longer than 15 minutes, and subsequently the assembly is washed several times with a wash solution, for example, with a saline solution such as phosphate buffered saline (PBS), optionally containing a detergent, for example T EEN-20®, buffered at a pH close to neutrality.
  • PBS phosphate buffered saline
  • the capture of the viral or subviral particles on the thermocycler tube can be performed by immunocapture by the use of molecules retained on said tube that have affinity for a component or structure related to said particle, for example, antibodies against proteins of the cover of the viral particle, or antibodies against proteins of the cover of a host cell containing the viral particle, or antibodies against double-stranded RNA when it is a viral or subviral particle.
  • the immobilization of molecules with affinity for said viral or subviral particle structures is first performed by any type of physical, chemical or chemical-physical interaction well known to those skilled in the art.
  • said molecules with affinity for a component of the viral or subviral particle are immobilized on the tube of the thermal cycler by adsorption, for which said tube is contacted with a solution of such molecules in a buffer at basic pH, the set is incubated at temperatures between 0 and 50 ° C, for a period of time normally greater than 15 minutes, and the set is washed several times with a wash solution, for example, a saline solution such as PBS, optionally containing a detergent, for example T EEN-20®, buffered at a pH close to neutrality.
  • a wash solution for example, a saline solution such as PBS, optionally containing a detergent, for example T EEN-20®, buffered at a pH close to neutrality.
  • the immobilized molecules on the tube of the thermal cycler are put in contact with the samples to be analyzed, the whole being incubated under the suitable temperature and time conditions so that said molecules immobilized on the tubes with affinity for such viral or sub-viral particles contained in the sample can immunocapture said particles.
  • the assembly is incubated at a temperature between 0 and 50 ° C, for a period of time normally greater than 15 minutes, followed by several washes with a washing solution, for example, as previously described (PBS + detergent ).
  • the second stage comprises the addition to the tubes of the thermal cycler, which contains the captured viral or subviral particles, of all the reagents necessary for the reverse transcription of the RNA contained in said captured particles and the enzymatic PCR amplification of a DNA fragment previously obtained.
  • the reagents necessary for the reverse transcription reaction comprise a reaction buffer, a reverse transcriptase, an initiating oligonucleotide, a ribonuclease inhibitor, deoxynucleoside triphosphate (dNTPs) and Mg 2+
  • the necessary reagents for the enzymatic amplification reaction by PCR they comprise a buffer for the reaction, a thermostable DNA polymerase, the direct and reverse initiating oligonucleotides, the dNTPs and Mg 2+ .
  • the reagents necessary to carry out the reverse transcription and enzymatic PCR amplification reactions generally comprise: a buffer suitable for performing both reactions;
  • the buffer may be any of the buffers commonly used in reverse transcription and enzymatic PCR amplification reactions, provided that said buffer is suitable for both reactions.
  • Reverse transcriptase can be any enzyme that catalyzes the synthesis of a copy DNA from an RNA that acts as a template.
  • said reverse transcriptase will only be active at the temperature at which the reverse transcription reaction is performed, and advantageously, the reverse transcriptase: (i) will be active at the temperature at which the reverse transcription reaction is performed, (ii ) it may be thermally inactivated and (iii) it will be inactive at temperatures at which the enzymatic amplification reaction is performed by PCR.
  • the reverse transcription reaction is carried out at a temperature equal to or less than 50 ° C, while the PCR enzymatic amplification reaction can be performed at both temperatures equal to or less than 50 ° C. as above 50 ° C, preferably at these latter temperatures. Therefore, in a specific embodiment of this invention the reverse transcriptase: (i) will be active at a temperature equal to or less than 50 ° C, (ii) will be thermally inactivated, and (iii) will be inactive at temperatures above 50 ° C .
  • thermostable DNA polymerase can be used to catalyze the synthesis of a DNA chain from a DNA that acts as a template, at an elevated temperature, such as Taq polymerase, Tfl polymerase, or thermostable DNA polymerases that are activated at temperatures higher than 90 ° C.
  • thermostable DNA polymerase is not active at the temperature at which the reverse transcription reaction is performed, and is only active at the temperature of carrying out the enzymatic PCR amplification reaction.
  • the reverse transcription reaction is typically carried out at a temperature equal to or less than 50 ° C, while the PCR enzymatic amplification reaction can be carried out both at temperatures equal to or below 50 ° C and above 50 ° C, preferably at these last temperatures. Therefore, in a specific embodiment of this invention the thermostable DNA polymerase will be inactive at temperatures equal to or less than 50 ° C.
  • An example of such an enzyme is that sold under the brand name AmpliTaq Gold® (Perkin-Elmer Cetus, United States).
  • an oligonucleotide complementary to an RNA fragment of the viral or subviral particle and of sufficient length for hybridization can be used, in a stable and specific manner, with said RNA fragment.
  • the inverse transcription reaction initiator is the same as the inverse (3 ') initiator that is used in the enzymatic PCR amplification reaction.
  • direct (5 1 ) and inverse (3 ') initiators can 12 appropriate oligonucleotides complementary to DNA sequences derived from the nucleic acid of the viral or subviral particle are used, with a length sufficient for hybridization, in a stable and specific manner, with said DNA sequences and allowing the polymerase to initiate synthesis of the complementary chain.
  • these primers will flank a specific, characteristic or identifying DNA fragment of the family, species or type, of the viral or subviral particle, so that the presence of said amplified fragment is indicative of the existence of the viral or subviral particle in question in the analyzed sample.
  • the reverse initiator (3 ') in the enzymatic PCR amplification reaction is the same as, and acts as, the initiator of the reverse transcription reaction.
  • RNA hydrolysis As a ribonuclease inhibitor, a product capable of inhibiting RNA hydrolysis can be used.
  • An example of such an inhibitor is the human placenta ribonuclease inhibitor (HPRI).
  • the dNTPs include deoxyadenosine triphosphate (dATP), deoxycytosin triphosphate (dCTP), deoxyguanidine triphosphate (dGTP) and deoxythymidine triphosphate (dTTP).
  • dATP deoxyadenosine triphosphate
  • dCTP deoxycytosin triphosphate
  • dGTP deoxyguanidine triphosphate
  • dTTP deoxythymidine triphosphate
  • MgCl 2 magnesium chloride
  • the reagents necessary for performing the reverse transcription and enzymatic PCR amplification steps comprise: a buffer suitable both for the reverse transcription reaction and for the reaction of 13 enzymatic amplification by PCR,
  • a reverse transcriptase activates at a temperature equal to or less than 50 ° C, thermally inactivatable, and inactive at temperatures above 50 ° C;
  • a thermostable DNA polymerase active at temperatures above 50 ° C and inactive at a temperature equal to or less than 50 ° C;
  • the common buffer for both reactions comprises 10 mM Tris-HCl, pH 8.3, 50 mM KC1, the MuLV reverse transcriptase, the human placenta ribonuclease inhibitor, an active DNA polymerase at temperatures above 50 ° C and inactive at temperatures between 37 and 50 ° C, MgCl 2 at a concentration between 1.5 and 5 mM, and a pair of oligonucleotide primers for enzymatic amplification by PCR flanking an identifying region of the viral particle or subviral, of which the reverse initiator oligonucleotide also acts as a reverse transcription initiator.
  • the reagents necessary for the reactions of reverse transcription and enzymatic amplification by PCR are commercial products or, in the case of the initiators, they are oligonucleotides with specific sequences that 14 can be synthesized by conventional techniques. These reagents can be added separately to the thermocycler tubes containing the captured particles or they can be pre-mixed with each other (all or some of them) and form a mixture or cocktail of reagents that is added to such tubes.
  • the third stage comprises introducing the tubes of the thermal cycler containing the viral or subviral particles captured together with the reagents necessary for the reactions of reverse transcription and enzymatic amplification by PCR, and applying to said tubes a suitable thermal cycle to perform First, the reverse transcription reaction, and after completion of this, the enzymatic PCR amplification reaction of the selected nucleic acid fragment, without removing the tube from the thermal cycler, without opening the tubes containing the test samples and with no more than modify the temperature
  • the thermal cycler is programmed to carry out a thermal cycle comprising: heating the thermocycler tubes at a temperature and for a period of time suitable for the reverse transcription reaction to take place, - heating the tubes at a temperature and during a suitable period of time to inactivate reverse transcriptase; Y
  • reverse transcription is performed at the operating temperature of the reverse transcriptase, which, in a particular embodiment of this invention will be equal to or less than 50 ° C, usually between 37 and 50 ° C, for a period of time comprised between 20 and 90 15 minutes .
  • the tubes are then heated to a temperature suitable for thermally denaturing the reverse transcriptase and, if using a thermostable DNA polymerase activatable at elevated temperature, activating said polymerase.
  • the tubes are heated to a temperature greater than 90 ° C, for a period of time between 2 and 10 minutes, whereby the reverse transcriptase is thermally inactivated, the DNA polymerase is activated (if any) and the RNA-DNA hybrid formed is denatured so that the PCR amplification reaction can begin.
  • the enzymatic PCR amplification reaction involves performing an adequate number of repeated heating and cooling cycles, each cycle comprising the steps of:
  • the banding temperature is equal to or greater than 60 ° C
  • the banding and elongation stages can be performed simultaneously.
  • the number of cycles to be performed depends on the degree of amplification of the nucleic acid fragment that is intended to be obtained. In general, adequate results are obtained with a number of cycles between 25 and 50. 16
  • said thermal cycle comprises: a) heating the reaction mixture, formed by the viral or subviral particles captured together with the reagents for the reactions of reverse transcription and enzymatic amplification by PCR, at a temperature between 37 and 48 ° C, for 30 minutes, in order to effect the reverse transcription reaction, then b) heat the reaction mixture at a temperature between 92 and 97 ° C, for 5 minutes, to inactivate the transcriptase conversely, denature the RNA-DNA hybrid and, where appropriate, activate the thermostable DNA polymerase; c) repeat a variable number of times, usually between 25 and 50 times, a heating and cooling cycle, comprising the steps of:
  • - denaturation normally at a temperature greater than 90 ° C, generally between 92 and 97 ° C, for a period of time greater than 15 seconds;
  • - banding at the hybridization temperature of the initiators to the template DNA;
  • - elongation at a temperature between approximately 60 and 94 ° C, for a period of time normally greater than 45 seconds; and d) at the end of the last cycle, maintain the elongation temperature for a period of time normally exceeding 2 minutes in order to terminate the PCR amplification reaction.
  • the fragments thus amplified can be subjected to analysis by spectrophotometric and / or techniques. 17 electrophoretic in order to detect, quantify and / or identify the viral or subviral particle in question, or alternatively they can be subjected to a structural analysis either by nucleic acid sequencing techniques (Maxam, AM, and Gilbert,., (1977 ), Proc. Nati. Acad. Sci. USA, 74: 560; and Sanger, F., et al., (1977), Proc. Nati. Acad. Sci.
  • PVY potato virus Y
  • Samples of potato leaves from healthy plants and plants infected with PVY are homogenized, separately, in a 1/10 ratio (weight / volume) with an extraction buffer composed of 0.5 M Tris-HCl, pH 8, 0, containing 2% polyvinyl pyrrolidone, 1% polyethylene glycol 6000, 0.8% NaCl, 0.005% T EEN-20® (SIGMA CHEMICAL CO., USA) and 0.02% sodium azide.
  • - dNTPs (dATP, dCTP, dGTP and dTTP), [2 ⁇ l of mixture of dNTPs at 10 mM of each one] (equivalent to 0.25 mM), - direct initiator (5 ') 2 ⁇ l to 10 ⁇ M ( 0.25 mM),
  • the initiators used are listed in the section on the LIST OF SEQUENCES, where the direct initiator (5 ') corresponds to the one identified as SEC. ID. N °: 1 and the inverse (3 ') to that identified as SEC. ID. N °: 2.
  • nt corresponding to the capsid protein gene plus the principle of the 3 'non-translatable region (3'-utr).
  • the direct initiator extends from nt 3869 to nt 3891 of the PVY genome, while the reverse initiator extends from nt 4745 to nt 4723.
  • the PVY genome sequence has been described by Hidaka et al., [ Nucleic Acid Research, 20, 3515, (1992)]. For the realization of this Example, 5 different concentrations of MgCl 2 (2, 2.25, 2.5, 2.75 and 3 mM) were tested for comparative purposes.
  • the tubes are then introduced into the thermal cycler where they undergo the following thermal cycle: a) heating at 42 ° C, for 30 minutes, with the 19 to carry out the reverse transcription reaction, b) heating at 95 ° C, for 5 minutes, to inactivate the reverse transcriptase and the ribonuclease inhibitor, activate the DNA polymerase, and denature the RNA-DNA hybrid, c) 35 cycles PCR comprising the steps of:
  • the amplified samples were subjected to 0.8% agarose gel electrophoresis in TAE buffer as indicated by Sambrook et al., (1989) [Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor ], were stained with a solution of ethidium bromide (4 ⁇ g / ⁇ l) and photographed with a Polaroid camera on an ultraviolet light transilluminator with an orange filter in front of the lens.
  • MuLV reverse transcriptase 50 units of MuLV reverse transcriptase (PERKIN-ELMER, USA) [1 ⁇ l of MuLV reverse transcriptase at 50 units / ⁇ l], - 20 units of HPRI (AMERSHAM, USA), [1 ⁇ l of HPRI at 200 units / ⁇ l ],
  • the tubes are introduced into the thermal cycler where they undergo the following thermal cycle: a) heating at 42 ° C, for 30 minutes, in order to carry out the reverse transcription reaction, b) heating at 97 ° C, for 5 minutes, to inactivate the reverse transcriptase and the ribonuclease inhibitor, and denature the RNA-DNA hybrid, 21 c) 35 cycles of PCR comprising the steps of:
  • the reverse transcription reaction is carried out in a final volume of 20 ⁇ l containing:
  • a buffer consisting of 10 mM Tris-HCl, pH 8.3, 50 mM KC1, - 5 mM MgCl 2 ,
  • MuLV reverse transcriptase 50 units of MuLV reverse transcriptase (PERKIN-ELMER, USA) and
  • the reverse initiator (3 ') used is the same as in Example 1.
  • the reverse transcription reaction was carried out by heating at 42 ° C for 30 minutes and subsequently 22 tubes were placed on ice.
  • the direct initiator (5 ') used is the same as in Example 1.
  • the tubes are introduced into the thermal cycler where they undergo the following thermal cycle: a) heating at 97 ° C, for 5 minutes, b) 35 PCR cycles comprising the steps of: - denaturation: 20 seconds at 94 ° C;
  • Example 2.1 The products resulting from the amplification reaction from both Example 2.1 and Example 2.2 were subjected to agarose gel electrophoresis as described in Example 1. The results obtained are shown in Figure 2, where it is observed that the yield obtained with the one-step reverse transcription-PCR reaction at a 3 mM concentration of MgCl 2 (Example 2.1: method provided by the invention) is several times higher than that obtained according to the procedure described in the patent 2. 3
  • Example 2.2 Spanish ES 9201232 (Example 2.2).
  • the greater brightness of the visible band in the lane corresponding to the amplified product according to Example 2.1 with a MgCl 2 concentration of 3 mM is indicative of the greater quantity of amplified product.

Abstract

The invention relates to a process which comprises capturing on a tube of a thermocycler the viral or sub-viral particles, adding into the tube the reagents which are necessary to carry out the reverse transcription (RT) and the enzymatic amplification by polymerase chain reaction (PCR), introducing the tube into the thermocycler and applying a thermocycle appropriate to effect first the reverse transcription and then the PCR without having to add reagents between both reactions and without having to manipulate the tubes with samples. To this effect, a thermostable DNA polymerase is used in the PCR. The amplified fragments can be subjected to various analyses for detecting, identifying and typing the viral or sub-viral particle. This process can be applied to diagnosis and studies of molecular epidemiology.

Description

1 one
PROCEDIMIENTO PARA LA OBTENCIÓN DE FRAGMENTOS AMPLIFICADOS DE ACIDO NUCLEICO DE PARTÍCULAS VIRALES Y SUBVIRALES QUE CONTIENEN RNAPROCEDURE FOR OBTAINING AMPLIFIED FRAGMENTS OF NUCLEIC ACID OF VIRAL AND SUBVIRAL PARTICLES CONTAINING RNA
CAMPO DE LA INVENCIÓNFIELD OF THE INVENTION
Esta invención se refiere a un procedimiento para la obtención de fragmentos amplificados de ácido nucleico de virus con genoma RNA y de partículas subvirales que contienen RNA. Los fragmentos amplificados resultantes pueden ser analizados posteriormente para detectar, identificar o tipificar molecularmente a dichos virus y partículas subvirales.This invention relates to a method for obtaining amplified fragments of virus nucleic acid with RNA genome and sub-viral particles containing RNA. The resulting amplified fragments can be subsequently analyzed to detect, identify or molecularly typify said viruses and subviral particles.
ANTECEDENTES DE LA INVENCIÓN La detección e identificación de patógenos virales y subvirales constituyen prácticas habituales en el control sanitario y en estudios epidemiológicos. Asimismo, el análisis estructural del genoma de patógenos virales y subvirales proporciona una valiosa información tanto para el diagnóstico, tipificación y epidemiología moleculares como en proyectos de diseño de epitopos para la obtención de vacunas y en programas de búsqueda de genes que confieran resistencia a dichos patógenos.BACKGROUND OF THE INVENTION The detection and identification of viral and subviral pathogens are common practices in health control and epidemiological studies. Likewise, the structural analysis of the genome of viral and subviral pathogens provides valuable information both for molecular diagnosis, typing and epidemiology, as well as for epitope design projects to obtain vaccines and in gene search programs that confer resistance to said pathogens. .
En todos los casos es necesario procesar, a bajo costo y sin ambigüedad, un elevado número de muestras durante cortos periodos de tiempo, por lo que es deseable que los procedimientos empleados sean sensibles, precisos, específicos, rápidos, susceptibles de automatización y que no requieran instalaciones, equipos y materiales ni complejos ni caros, ni operadores con alta preparación técnica .In all cases it is necessary to process, at low cost and without ambiguity, a large number of samples for short periods of time, so it is desirable that the procedures used are sensitive, precise, specific, fast, susceptible to automation and not require facilities, equipment and materials neither complex nor expensive, nor operators with high technical preparation.
Se conocen métodos para la detección, identificación y tipificación molecular de patógenos virales y subvirales basados en el análisis de fragmentos amplificados mediante la reacción en cadena de la polimerasa (PCR) . En general, 2 tales métodos comprenden la obtención del fragmento amplificado de ácido nucleico y el análisis posterior del mismo. El empleo de técnicas espectrofotométricas y/o electroforéticas para el análisis de dicho fragmento conduce a la detección -y/o identificación del patógeno, mientras que el análisis estructural del fragmento amplificado bien por técnicas de secuenciación, o bien por técnicas de análisis de polimorfismos de longitud de fragmentos de restricción (RFLP) o de conformación de- cadena sencilla (SSCP) , permite la tipificación molecular del patógeno y la realización de estudios de epidemiología molecular.Methods for the detection, identification and molecular typing of viral and subviral pathogens based on the analysis of amplified fragments by polymerase chain reaction (PCR) are known. Usually, 2 such methods comprise obtaining the amplified nucleic acid fragment and subsequent analysis thereof. The use of spectrophotometric and / or electrophoretic techniques for the analysis of said fragment leads to the detection and / or identification of the pathogen, while the structural analysis of the fragment amplified either by sequencing techniques or by polymorphism analysis techniques of Length of restriction fragments (RFLP) or single chain conformation (SSCP), allows the molecular typing of the pathogen and the conduct of molecular epidemiology studies.
Algunos métodos para la obtención de fragmentos amplificados de DNA combinan la captura del patógeno con la amplificación enzimática por PCR. En el caso de patógenos virales o subvirales con genoma RNA, la amplificación por PCR va precedida de una etapa de transcripción inversa con el fin de convertir el RNA en DNA (sustrato para la PCR) . Se conocen unos métodos que incluyen la realización de una etapa de lisis de la partícula viral capturada y liberación del ácido nucleico viral antes de la realización de la amplificación enzimática o, en su caso, de la transcripción inversa (por ejemplo, patente norteamericana US 5.077.192), mientras que otros métodos prescinden de dicha etapa de lisis y liberación previa de ácido nucleico (patentes españolas ES 9201232 y ES 9302657) .Some methods for obtaining amplified DNA fragments combine the capture of the pathogen with the enzymatic amplification by PCR. In the case of viral or subviral pathogens with RNA genome, PCR amplification is preceded by a reverse transcription step in order to convert RNA to DNA (substrate for PCR). Methods are known that include performing a step of lysis of the captured viral particle and release of the viral nucleic acid before performing the enzyme amplification or, where appropriate, reverse transcription (e.g., US Patent 5,077). .192), while other methods dispense with said stage of lysis and prior release of nucleic acid (Spanish patents ES 9201232 and ES 9302657).
En general, la obtención de un fragmento amplificado de ácido nucleico según la metodología descrita en dichas patentes españolas comprende: (a) la inmunocaptura del patógeno en los pocilios de una placa de microtitulación,In general, obtaining an amplified nucleic acid fragment according to the methodology described in said Spanish patents comprises: (a) the immunocapture of the pathogen in the wells of a microtiter plate,
(b) la transcripción inversa del RNA viral o subviral, sin lisis ni desnaturalización previa de la partícula inmunocapturada, en la placa de microtitulación, mediante la adición de los reactivos necesarios para la realización de dicha reacción, y(b) reverse transcription of the viral or subviral RNA, without lysis or prior denaturation of the immunocaptured particle, in the microtiter plate, by adding the reagents necessary for the realization of said reaction, and
(c) la amplificación enzimática por PCR de un fragmento de ácido nucleico del patógeno después de que haya finalizado la transcripción inversa y tras la adición de los reactivos necesarios para realizar dicha reacción de amplificación .(c) the enzymatic PCR amplification of a nucleic acid fragment of the pathogen after the reverse transcription is complete and after the addition of the reagents necessary to perform said amplification reaction.
La obtención de fragmentos amplificados de ácido nucleico de un patógeno según la metodología descrita en las patentes españolas ES 9201232 y ES 9302657 antes citadas, presenta algunos inconvenientes, relacionados con:Obtaining amplified nucleic acid fragments of a pathogen according to the methodology described in Spanish patents ES 9201232 and ES 9302657 cited above, presents some drawbacks, related to:
- el empleo de un volumen de RNA viral inicial pequeño, típicamente 20 μl, para realizar la transcripción inversa; y- the use of a small initial viral RNA volume, typically 20 μl, to perform reverse transcription; Y
- la realización de la amplificación enzimática por PCR una vez que ha terminado la reacción de transcripción inversa y se han añadido los reactivos necesarios para dicha reacción de amplificación implica la realización de una serie de operaciones, tales como, destapar los pocilios, añadir los reactivos necesarios a cada pocilio y volver a taparlos, lo que lleva consigo no sólo (a) un consumo de tiempo y un aumento en el número de operaciones de manipulación a realizar sobre las muestras, especialmente importante en estudios de epidemiología donde se debe procesar un número extraordinariamente alto de muestras, lo que limita considerablemente la capacidad de automatismo de dicho método, sino que, además, (b) existe un cierto riesgo de una posible contaminación de las muestras por contacto accidental del contenido de un pocilio en otro o por efecto del entorno ambiental, lo que puede incidir negativamente en la fiabilidad y reproducibilidad del método.- the realization of the enzymatic amplification by PCR once the reverse transcription reaction has ended and the necessary reagents for said amplification reaction have been added implies the performance of a series of operations, such as uncovering the wells, adding the reagents necessary to each well and to cover them again, which entails not only (a) a consumption of time and an increase in the number of handling operations to be performed on the samples, especially important in epidemiology studies where a number must be processed extraordinarily high in samples, which considerably limits the automatic capacity of said method, but, in addition, (b) there is a certain risk of possible contamination of the samples by accidental contact of the contents of one well in another or by the effect of environmental environment, which can have a negative impact on the reliability and reproducibility of the method.
Por consiguiente, sigue existiendo la necesidad de disponer de un método para la obtención de fragmentos amplificados de ácido nucleico de partículas virales y subvirales que contienen RNA que supere los inconvenientes previamente mencionados. En particular, seria muy conveniente que dicho método permitiera llevar a cabo la transcripción inversa en presencia de mayor volumen de la solución que contiene el RNA viral inicial, disminuyera el riesgo de contaminaciones, redujera el número de operaciones a realizar con las muestras, facilitara el manejo de las mismas y fuera susceptible de automatismo. Estos objetivos se cumplen con el método propuesto por la presente invención.Therefore, there remains a need for a method for obtaining amplified fragments of nucleic acid from viral and sub-viral particles that contain RNA that overcomes the drawbacks. previously mentioned. In particular, it would be very convenient if such a method allowed reverse transcription to be carried out in the presence of a larger volume of the solution containing the initial viral RNA, decreased the risk of contamination, reduced the number of operations to be performed with the samples, facilitated the handling them and being susceptible to automatism. These objectives are met with the method proposed by the present invention.
COMPENDIO DE LA INVENCIÓNSUMMARY OF THE INVENTION
La presente invención proporciona una solución a los problemas planteados previamente que consiste en un procedimiento para la obtención de fragmentos amplificados de ácido nucleico de partículas virales o subvirales que contienen RNA, que combina (a) la captura del patógeno con (b) la adición, en una sola operación, de todos los reactivos que intervienen en la reacción de transcripción inversa y en la reacción de amplificación enzimática por PCR, y con (c) el desarrollo de unos ciclos térmicos apropiados para realizar, en primer lugar, la reacción de transcripción inversa y, en segundo lugar, sin necesidad de destapar el recipiente que contiene la muestra a ensayar y sin necesidad de añadir ningún reactivo adicional, sino simplemente modificando la temperatura, efectuar la reacción de amplificación enzimática por PCR del fragmento de ácido nucleico.The present invention provides a solution to the previously raised problems consisting of a process for obtaining amplified fragments of nucleic acid from viral or subviral particles containing RNA, which combines (a) the capture of the pathogen with (b) the addition, in a single operation, of all the reagents involved in the reverse transcription reaction and in the enzymatic amplification reaction by PCR, and with (c) the development of appropriate thermal cycles to perform, in the first place, the transcription reaction Inverse, and secondly, without the need to uncover the container containing the sample to be tested and without the need to add any additional reagents, but simply by modifying the temperature, carry out the PCR enzymatic amplification reaction of the nucleic acid fragment.
El fragmento amplificado puede ser sometido a técnicas espectrofotométricas, electroforéticas, o bien a análisis estructural para detectar, cuantificar, identificar o tipificar molecularmente la partícula viral o subviral en cuestión.The amplified fragment can be subjected to spectrophotometric, electrophoretic techniques, or to structural analysis to detect, quantify, identify or molecularly typify the viral or subviral particle in question.
Este método permite el empleo de un mayor volumen de la solución que contiene el RNA viral inicial para realizar la transcripción inversa (típicamente 80 μl) , es decir, se parte de mayor cantidad de muestra y el rendimiento y la sensibilidad aumentan, asimismo se disminuye el riesgo de contaminaciones accidentales, se reduce el número de operaciones de manipulación de las muestras, se facilita su automatización y permite aumentar el número de muestras a manejar.This method allows the use of a larger volume of the solution containing the initial viral RNA to perform reverse transcription (typically 80 μl), that is, A larger part of the sample and the yield and sensitivity increase, the risk of accidental contamination is reduced, the number of sample handling operations is reduced, automation is facilitated and the number of samples to be handled can be increased.
BREVE DESCRIPCIÓN DE LAS FIGURASBRIEF DESCRIPTION OF THE FIGURES
La Figura 1 muestra una fotografía de un gel de electroforesis donde se observa la presencia de un fragmento de 879 nucleótidos del genoma del virus Y de la patata (PVY), que corresponde al fragmento amplificado según el procedimiento proporcionado por esta invención y descrito en el Ejemplo 1. La Figura 2 muestra una fotografía de un gel de electroforesis donde se observa la presencia de un fragmento de 879 nucleótidos del genoma del PVY, que corresponde al fragmento amplificado según el procedimiento descrito en el Ejemplo 1, comparado con el mismo fragmento obtenido siguiendo el procedimiento descrito en la patente española ES 9201232.Figure 1 shows a photograph of an electrophoresis gel showing the presence of an 879 nucleotide fragment of the potato Y virus genome (PVY), corresponding to the amplified fragment according to the procedure provided by this invention and described in the Example 1. Figure 2 shows a photograph of an electrophoresis gel showing the presence of an 879 nucleotide fragment of the PVY genome, corresponding to the amplified fragment according to the procedure described in Example 1, compared to the same fragment obtained following the procedure described in Spanish patent ES 9201232.
DESCRIPCIÓN DETALLADA DE LA INVENCIÓNDETAILED DESCRIPTION OF THE INVENTION
El procedimiento para la obtención de fragmentos amplificados de ácido nucleico de partículas virales o subvirales que contienen RNA presentes en una muestra sospechosa de contener tales partículas, objeto de esta invención, comprende las etapas de: a) añadir dicha muestra a los tubos de un termociclador e incubar el conjunto bajo condiciones adecuadas para capturar dichas partículas virales o subvirales en los tubos del termociclador; b) retirar el sobrenadante de los tubos y lavar el conjunto de partículas virales o subvirales capturadas en los tubos del termociclador; c) añadir a dichos tubos, que contienen las partículas capturadas, todos los reactivos necesarios para realizar la reacción de transcripción inversa y la reacción de amplificación enzimática por PCR; d) introducir los tubos en el termociclador; y e) programar en el termociclador un ciclo térmico adecuado para que tenga lugar, en cada tubo, en primer lugar, la reacción de transcripción inversa del RNA, y posteriormente, la reacción de amplificación enzimática por PCR del fragmento de ácido nucleico tras desactivación térmica de la transcriptasa inversa, para lo cual dicho ciclo térmico comprende: calentar los tubos del termociclador a una temperatura y durante un periodo de tiempo adecuados para que tenga lugar la reacción de transcripción inversa, seguido deThe method for obtaining amplified nucleic acid fragments of viral or subviral particles containing RNA present in a sample suspected of containing such particles, object of this invention, comprises the steps of: a) adding said sample to the tubes of a thermal cycler and incubating the assembly under suitable conditions to capture said viral or subviral particles in the thermocycler tubes; b) remove the supernatant from the tubes and wash the set of viral or sub-viral particles captured in the tubes of the thermal cycler; c) adding to said tubes, which contain the captured particles, all the reagents necessary to carry out the reverse transcription reaction and the enzymatic amplification reaction by PCR; d) insert the tubes into the thermal cycler; and e) program a suitable thermal cycle in the thermal cycler so that the reverse transcription reaction of the RNA takes place in each tube first, and subsequently the enzymatic PCR amplification reaction of the nucleic acid fragment after thermal deactivation of the reverse transcriptase, for which said thermal cycle comprises: heating the thermocycler tubes at a temperature and for a suitable period of time for the reverse transcription reaction to take place, followed by
- calentar los tubos a una temperatura y durante un periodo de tiempo adecuados para inactivar la transcriptasa inversa; - realizar un número variable y repetido de ciclos de PCR a las temperaturas y tiempos de desnaturalización, anillamiento y elongación adecuados.- heating the tubes at a temperature and for a suitable period of time to inactivate the reverse transcriptase; - perform a variable and repeated number of PCR cycles at the appropriate temperatures and times of denaturation, banding and elongation.
De forma más concreta y para la puesta en práctica del procedimiento objeto de esta invención, a continuación, se describen las distintas etapas que lo componen.More specifically and for the implementation of the process object of this invention, the different stages that compose it are described below.
A) Captura de la partícula La primera etapa del procedimiento comprende la captura de la partícula viral o subviral que contiene RNA en los tubos de un termociclador.A) Particle capture The first stage of the procedure involves the capture of the viral or subviral particle that contains RNA in the tubes of a thermal cycler.
El término "partícula viral", en el sentido utilizado en esta descripción, incluye a cualquier virus con genomaThe term "viral particle", in the sense used in this description, includes any genome virus.
RNA que se replique en el interior de protoplastos, células eucarióticas o procarióticas, y a expensas de algún enzima de dichos protoplastos o células, por ejemplo, virus 7 animales, virus de plantas, virus bacterianos y virus fúngicos. El término "partícula subviral", tal como aquí se utiliza, incluye viroides, virusoides y virus satélites, con genoma RNA, que se repliquen en el interior de células vegetales, o de sus protoplastos y a expensas de algún enzima de dichos protoplastos o células. Un caso particular de tales partículas virales o subvirales son los patógenos virales o subvirales, término en el que se incluyen los virus con genoma RNA y las partículas subvirales que comprenden RNA que pueden ocasionar cualquier patología tanto en animales, incluido el hombre, como en plantas y hongos .RNA that replicates inside protoplasts, eukaryotic or prokaryotic cells, and at the expense of some enzyme of said protoplasts or cells, for example, viruses 7 animals, plant viruses, bacterial viruses and fungal viruses. The term "subviral particle", as used herein, includes viroids, virusoids and virus satellites, with RNA genome, which replicate inside plant cells, or their protoplasts and at the expense of some enzyme of said protoplasts or cells. A particular case of such viral or sub-viral particles is viral or sub-viral pathogens, a term in which viruses with an RNA genome are included and sub-viral particles that comprise RNA that can cause any pathology in animals, including man, or in plants and mushrooms.
El tubo donde se pueden capturar las partículas virales o subvirales es un tubo convencional, de los usados en los termocicladores habitualmente utilizados para llevar a cabo la amplificación enzimática de fragmentos de DNA por PCR.The tube where the viral or sub-viral particles can be captured is a conventional tube, of those used in the thermal cyclers commonly used to carry out the enzymatic amplification of DNA fragments by PCR.
En general, las partículas virales o subvirales se encontrarán en una muestra que puede ser medioambiental, por ejemplo, una muestra de agua o de aire, o una muestra biológica, cuyo término incluye células eucarióticas, procarióticas, protoplastos, micelios, tejidos y órganos animales y vegetales, medios de cultivo in vi tro de cualquiera de las entidades anteriores, sistemas vegetales o animales, organismos vegetales o animales, así como cualquier producto de excreción, secreción o transformación de cualquiera de las entidades anteriores.In general, viral or subviral particles will be found in a sample that may be environmental, for example, a water or air sample, or a biological sample, the term of which includes eukaryotic, prokaryotic, protoplasts, mycelium, animal tissues and organs and vegetables, in vitro culture media of any of the above entities, plant or animal systems, plant or animal organisms, as well as any excretion, secretion or transformation product of any of the above entities.
Para efectuar la captura de las partículas virales o subvirales, la muestra susceptible de contenerlas, que previamente ha sido homogeneizada, en su caso, por ejemplo, en un tampón adecuado, se añade al tubo del termociclador, incubando el conjunto a la temperatura y durante el tiempo adecuados que permiten la captura de dichas partículas. En general, el conjunto tubo-muestra se incuba a temperaturas comprendidas entre 0 y 50°C, durante un periodo de tiempo 8 normalmente superior a 15 minutos, y posteriormente el conjunto se lava varias veces con una solución de lavado, por ejemplo, con una solución salina tal como tampón fosfato salino (PBS) , opcionalmente conteniendo un detergente, por ejemplo T EEN-20®, tamponada a un pH próximo a la neutralidad.In order to capture the viral or sub-viral particles, the sample capable of containing them, which has previously been homogenized, where appropriate, for example, in a suitable buffer, is added to the tube of the thermal cycler, incubating the assembly at the temperature and during adequate time allowing the capture of said particles. In general, the tube-sample set is incubated at temperatures between 0 and 50 ° C, for a period of time 8 normally longer than 15 minutes, and subsequently the assembly is washed several times with a wash solution, for example, with a saline solution such as phosphate buffered saline (PBS), optionally containing a detergent, for example T EEN-20®, buffered at a pH close to neutrality.
Alternativamente, la captura de las partículas virales o subvirales sobre el tubo del termociclador puede efectuarse por inmunocaptura mediante el empleo de moléculas retenidas sobre dicho tubo que tengan afinidad por un componente o estructura relacionada con dicha partícula, por ejemplo, anticuerpos frente a proteínas de la cubierta de la partícula viral, o anticuerpos frente a proteínas de la cubierta de una célula hospedadora que contenga la partícula viral, o anticuerpos frente a RNA de doble cadena cuando se trate de una partícula viral o subviral. En esta alternativa, en primer lugar se realiza la inmovilización de las moléculas con afinidad por dichas estructuras de la partícula viral o subviral mediante cualquier tipo de interacción fisica, química o químico- física bien conocida por los expertos en esta materia. En una realización particular, dichas moléculas con afinidad por un componente de la partícula viral o subviral se inmovilizan sobre el tubo del termociclador por adsorción, para lo cual dicho tubo se pone en contacto con una solución de tales moléculas en un tampón a pH básico, incubándose el conjunto a temperaturas comprendidas entre 0 y 50°C, durante un periodo de tiempo normalmente superior a 15 minutos, y el conjunto se lava varias veces con una solución de lavado, por ejemplo, una solución salina tal como PBS, opcionalmente conteniendo un detergente, por ejemplo T EEN-20®, tamponada a un pH próximo a la neutralidad. A continuación, las moléculas inmovilizadas sobre el tubo del termociclador se ponen en contacto con las muestras a analizar, incubándose el conjunto bajo las condiciones de temperatura y tiempo adecuadas para que dichas moléculas inmovilizadas sobre los tubos con afinidad por tales partículas virales o subvirales contenidas en la muestra puedan inmunocapturar dichas partículas. En general, el conjunto se incuba a una temperatura comprendida entre 0 y 50°C, durante un periodo de tiempo normalmente superior a 15 minutos, seguido de varios lavados con una solución de lavado, por ejemplo, como la descrita previamente (PBS + detergente) .Alternatively, the capture of the viral or subviral particles on the thermocycler tube can be performed by immunocapture by the use of molecules retained on said tube that have affinity for a component or structure related to said particle, for example, antibodies against proteins of the cover of the viral particle, or antibodies against proteins of the cover of a host cell containing the viral particle, or antibodies against double-stranded RNA when it is a viral or subviral particle. In this alternative, the immobilization of molecules with affinity for said viral or subviral particle structures is first performed by any type of physical, chemical or chemical-physical interaction well known to those skilled in the art. In a particular embodiment, said molecules with affinity for a component of the viral or subviral particle are immobilized on the tube of the thermal cycler by adsorption, for which said tube is contacted with a solution of such molecules in a buffer at basic pH, the set is incubated at temperatures between 0 and 50 ° C, for a period of time normally greater than 15 minutes, and the set is washed several times with a wash solution, for example, a saline solution such as PBS, optionally containing a detergent, for example T EEN-20®, buffered at a pH close to neutrality. Next, the immobilized molecules on the tube of the thermal cycler are put in contact with the samples to be analyzed, the whole being incubated under the suitable temperature and time conditions so that said molecules immobilized on the tubes with affinity for such viral or sub-viral particles contained in the sample can immunocapture said particles. In general, the assembly is incubated at a temperature between 0 and 50 ° C, for a period of time normally greater than 15 minutes, followed by several washes with a washing solution, for example, as previously described (PBS + detergent ).
B) Adición de los reactivos La segunda etapa comprende la adición a los tubos del termociclador, que contienen las partículas virales o subvirales capturadas, de todos los reactivos necesarios para la transcripción inversa del RNA contenido en dichas partículas capturadas y la amplificación enzimática por PCR de un fragmento del DNA previamente obtenido.B) Addition of the reagents The second stage comprises the addition to the tubes of the thermal cycler, which contains the captured viral or subviral particles, of all the reagents necessary for the reverse transcription of the RNA contained in said captured particles and the enzymatic PCR amplification of a DNA fragment previously obtained.
En general, los reactivos necesarios para la reacción de transcripción inversa comprenden un tampón para la reacción, una transcriptasa inversa, un oligonucleótido iniciador, un inhibidor de ribonucleasas, los desoxi- nucleósidos trifosfato (dNTPs) y Mg2+, mientras que los reactivos necesarios para la reacción de amplificación enzimática por PCR comprenden un tampón para la reacción, una DNA polimerasa termoestable, los oligonucleótidos iniciadores directo e inverso, los dNTPs y Mg2+.In general, the reagents necessary for the reverse transcription reaction comprise a reaction buffer, a reverse transcriptase, an initiating oligonucleotide, a ribonuclease inhibitor, deoxynucleoside triphosphate (dNTPs) and Mg 2+ , while the necessary reagents for the enzymatic amplification reaction by PCR they comprise a buffer for the reaction, a thermostable DNA polymerase, the direct and reverse initiating oligonucleotides, the dNTPs and Mg 2+ .
Por consiguiente, los reactivos necesarios para llevar a cabo las reacciones de transcripción inversa y amplificación enzimática por PCR comprenden, en general: - un tampón adecuado para la realización de ambas reacciones;Accordingly, the reagents necessary to carry out the reverse transcription and enzymatic PCR amplification reactions generally comprise: a buffer suitable for performing both reactions;
- una transcriptasa inversa;- a reverse transcriptase;
- una DNA polimerasa termoestable;- a thermostable DNA polymerase;
- un oligonucleótido iniciador para la reacción de transcripción inversa; 10- an initiating oligonucleotide for the reverse transcription reaction; 10
- un par de oligonucleótidos iniciadores directo e inverso para la reacción de amplificación enzimática por PCR;- a pair of direct and reverse oligonucleotide primers for the enzymatic amplification reaction by PCR;
- un inhibidor de ribonucleasas; - los dNTPs; y- a ribonuclease inhibitor; - dNTPs; Y
- Mg+.- Mg + .
El tampón puede ser cualquiera de los tampones habitualmente utilizados en las reacciones de transcripción inversa y de amplificación enzimática por PCR, siempre que dicho tampón resulte adecuado para realizar ambas reacciones .The buffer may be any of the buffers commonly used in reverse transcription and enzymatic PCR amplification reactions, provided that said buffer is suitable for both reactions.
La transcriptasa inversa (DNA polimerasa dependiente de RNA) puede ser cualquier enzima que catalice la síntesis de un DNA copia a partir de un RNA que actúe como molde. Preferentemente, dicha transcriptasa inversa será sólo activa a la temperatura en la que se realice la reacción de transcripción inversa, y ventajosamente, la transcriptasa inversa: (i) será activa a la temperatura en la que se realiza la reacción de transcripción inversa, (ii) se podrá inactivar térmicamente y (iii) será inactiva a temperaturas a las que se realiza la reacción de amplificación enzimática por PCR. En una realización particular de esta invención, la reacción de transcripción inversa se lleva a cabo a una temperatura igual o inferior a 50°C, mientras que la reacción de amplificación enzimática por PCR se puede realizar tanto a temperaturas iguales o inferiores a 50°C como superiores a 50°C, preferentemente a estas últimas temperaturas. Por tanto, en una realización concreta de esta invención la transcriptasa inversa: (i) será activa a una temperatura igual o inferior a 50°C, (ii) se inactivará térmicamente, y (iii) será inactiva a temperaturas superiores a 50°C. Ejemplos de dicha enzima son la transcriptasa inversa del virus de la mieloblastosis de ave (AMV) , activa a temperaturas comprendidas entre 37 y 48°C, y la transcriptasa inversa del virus de la leucemia 11 murina (MuLV) , activa a temperaturas comprendidas entre 37 y 42°C. Ambas enzimas se pueden inactivar irreversiblemente mediante un tratamiento térmico a temperaturas superiores a los 90°C. Como DNA polimerasa termoestable puede utilizarse cualquier enzima que catalice la síntesis de una cadena de DNA a partir de un DNA que actúe como molde, a temperatura elevada, tal como la polimerasa Taq, la polimerasa Tfl, o DNA polimerasas termoestables que se activan a temperaturas superiores a los 90°C. En una realización particular y preferida, dicha DNA polimerasa termoestable no es activa a la temperatura en la que se realiza la reacción de transcripción inversa, y sólo es activa a la temperatura de realización de la reacción de amplificación enzimática por PCR. La reacción de transcripción inversa se lleva a cabo típicamente a una temperatura igual o inferior a 50°C, mientras que la reacción de amplificación enzimática por PCR se puede realizar tanto a temperaturas iguales o inferiores a 50°C como superiores a 50°C, preferentemente a estas últimas temperaturas. Por tanto, en una realización concreta de esta invención la DNA polimerasa termoestable será inactiva a temperaturas iguales o inferiores a 50°C. Un ejemplo de dicha enzima es la comercializada con la marca AmpliTaq Gold® (Perkin-Elmer Cetus, Estados Unidos) . Como iniciador de la reacción de transcripción inversa se puede utilizar un oligonucleótido complementario a un fragmento de RNA de la partícula viral o subviral y de una longitud suficiente para su hibridación, de forma estable y especifica, con dicho fragmento de RNA. En una realización particular y preferida de esta invención, para evitar amplificaciones indeseables, el iniciador de la reacción de transcripción inversa es el mismo que el iniciador inverso (3') que se utiliza en la reacción de amplificación enzimática por PCR. Como iniciadores directo (51) e inverso (3') pueden 12 utilizarse unos oligonucleótidos apropiados complementarios a unas secuencias de DNA derivadas del ácido nucleico de la partícula viral o subviral, con una longitud suficiente para su hibridación, de forma estable y específica, con dichas secuencias de DNA y que permiten a la polimerasa iniciar la síntesis de la cadena complementaria. En una realización preferida de esta invención, estos iniciadores flanquearán un fragmento de DNA especifico, característico o identificativo de la familia, especie o tipo, de la partícula viral o subviral, de forma que la presencia de dicho fragmento amplificado es indicativo de la existencia de la partícula viral o subviral en cuestión en la muestra analizada. Como se ha mencionado previamente, en una realización particular y preferida de esta invención el iniciador inverso (3') en la reacción de amplificación enzimática por PCR es el mismo que, y actúa como, el iniciador de la reacción de transcripción inversa.Reverse transcriptase (RNA-dependent DNA polymerase) can be any enzyme that catalyzes the synthesis of a copy DNA from an RNA that acts as a template. Preferably, said reverse transcriptase will only be active at the temperature at which the reverse transcription reaction is performed, and advantageously, the reverse transcriptase: (i) will be active at the temperature at which the reverse transcription reaction is performed, (ii ) it may be thermally inactivated and (iii) it will be inactive at temperatures at which the enzymatic amplification reaction is performed by PCR. In a particular embodiment of this invention, the reverse transcription reaction is carried out at a temperature equal to or less than 50 ° C, while the PCR enzymatic amplification reaction can be performed at both temperatures equal to or less than 50 ° C. as above 50 ° C, preferably at these latter temperatures. Therefore, in a specific embodiment of this invention the reverse transcriptase: (i) will be active at a temperature equal to or less than 50 ° C, (ii) will be thermally inactivated, and (iii) will be inactive at temperatures above 50 ° C . Examples of said enzyme are the reverse transcriptase of the bird myeloblastosis virus (AMV), active at temperatures between 37 and 48 ° C, and the reverse transcriptase of the leukemia virus 11 murine (MuLV), active at temperatures between 37 and 42 ° C. Both enzymes can be irreversibly inactivated by heat treatment at temperatures above 90 ° C. Any thermostable DNA polymerase can be used to catalyze the synthesis of a DNA chain from a DNA that acts as a template, at an elevated temperature, such as Taq polymerase, Tfl polymerase, or thermostable DNA polymerases that are activated at temperatures higher than 90 ° C. In a particular and preferred embodiment, said thermostable DNA polymerase is not active at the temperature at which the reverse transcription reaction is performed, and is only active at the temperature of carrying out the enzymatic PCR amplification reaction. The reverse transcription reaction is typically carried out at a temperature equal to or less than 50 ° C, while the PCR enzymatic amplification reaction can be carried out both at temperatures equal to or below 50 ° C and above 50 ° C, preferably at these last temperatures. Therefore, in a specific embodiment of this invention the thermostable DNA polymerase will be inactive at temperatures equal to or less than 50 ° C. An example of such an enzyme is that sold under the brand name AmpliTaq Gold® (Perkin-Elmer Cetus, United States). As an initiator of the reverse transcription reaction, an oligonucleotide complementary to an RNA fragment of the viral or subviral particle and of sufficient length for hybridization can be used, in a stable and specific manner, with said RNA fragment. In a particular and preferred embodiment of this invention, to avoid undesirable amplifications, the inverse transcription reaction initiator is the same as the inverse (3 ') initiator that is used in the enzymatic PCR amplification reaction. As direct (5 1 ) and inverse (3 ') initiators can 12 appropriate oligonucleotides complementary to DNA sequences derived from the nucleic acid of the viral or subviral particle are used, with a length sufficient for hybridization, in a stable and specific manner, with said DNA sequences and allowing the polymerase to initiate synthesis of the complementary chain. In a preferred embodiment of this invention, these primers will flank a specific, characteristic or identifying DNA fragment of the family, species or type, of the viral or subviral particle, so that the presence of said amplified fragment is indicative of the existence of the viral or subviral particle in question in the analyzed sample. As previously mentioned, in a particular and preferred embodiment of this invention the reverse initiator (3 ') in the enzymatic PCR amplification reaction is the same as, and acts as, the initiator of the reverse transcription reaction.
Como inhibidor de ribonucleasas puede utilizarse un producto capaz de inhibir la hidrólisis del RNA. Un ejemplo de dicho inhibidor es el inhibidor de ribonucleasas de placenta humana (HPRI) .As a ribonuclease inhibitor, a product capable of inhibiting RNA hydrolysis can be used. An example of such an inhibitor is the human placenta ribonuclease inhibitor (HPRI).
Los dNTPs incluyen desoxiadenosin-trifosfato (dATP) , desoxicitosin-trifosfato (dCTP) , desoxiguanidin-trifosfato (dGTP) y desoxitimidin-trifosfato (dTTP) . Como fuente de Mg2+ puede añadirse cloruro magnésico (MgCl2) . Se ha observado que la presencia de este ion es necesaria para realizar eficazmente las reacciones de transcripción inversa y de amplificación enzimática por PCR en general. Por tanto, en una realización particular de esta invención, los reactivos necesarios para la realización de las etapas de transcripción inversa y amplificación enzimática por PCR comprenden: un tampón adecuado tanto para la reacción de transcripción inversa como para la reacción de 13 amplificación enzimática por PCR,The dNTPs include deoxyadenosine triphosphate (dATP), deoxycytosin triphosphate (dCTP), deoxyguanidine triphosphate (dGTP) and deoxythymidine triphosphate (dTTP). As the source of Mg 2+, magnesium chloride (MgCl 2 ) can be added. It has been observed that the presence of this ion is necessary to effectively perform the reverse transcription and enzymatic amplification reactions by PCR in general. Therefore, in a particular embodiment of this invention, the reagents necessary for performing the reverse transcription and enzymatic PCR amplification steps comprise: a buffer suitable both for the reverse transcription reaction and for the reaction of 13 enzymatic amplification by PCR,
- una transcriptasa inversa activa a una temperatura igual o inferior a 50°C, inactivable térmicamente, e inactiva a temperaturas superiores a 50°C; - una DNA polimerasa termoestable, activa a temperaturas superiores a 50°C e inactiva a una temperatura igual o inferior a 50°C;- a reverse transcriptase activates at a temperature equal to or less than 50 ° C, thermally inactivatable, and inactive at temperatures above 50 ° C; - a thermostable DNA polymerase, active at temperatures above 50 ° C and inactive at a temperature equal to or less than 50 ° C;
- un oligonucleótido adecuado como iniciador para la reacción de transcripción inversa y como iniciador inverso (3') del par de oligonucleótidos de la reacción de amplificación enzimática por PCR;- an oligonucleotide suitable as an initiator for the reverse transcription reaction and as a reverse initiator (3 ') of the oligonucleotide pair of the PCR amplification reaction;
- un oligonucleótido directo (5') para la reacción de amplificación enzimática por PCR, que junto con el iniciador inverso (3') forman un par de oligonucleótidos que flanquea una región que permite la identificación y caracterización de la partícula viral o subviral;- a direct oligonucleotide (5 ') for the enzymatic amplification reaction by PCR, which together with the reverse initiator (3') form a pair of oligonucleotides flanking a region that allows the identification and characterization of the viral or subviral particle;
- un inhibidor de ribonucleasas;- a ribonuclease inhibitor;
- dATP, dCTP, dGTP y dTTP; y- dATP, dCTP, dGTP and dTTP; Y
- Mg2+. En una realización particular, el tampón común para ambas reacciones comprende Tris-HCl 10 mM, pH 8,3, KC1 50 mM, la transcriptasa inversa de MuLV, el inhibidor de ribonucleasas de placenta humana, una DNA polimerasa activa a temperaturas superiores a 50°C e inactiva a temperaturas comprendidas entre 37 y 50°C, MgCl2 en una concentración comprendida entre 1,5 y 5 mM, y un par de oligonucleótidos iniciadores para la amplificación enzimática por PCR que flanquean una región identificativa de la partícula viral o subviral, de los cuales, el oligonucleótido iniciador inverso actúa también como iniciador de la transcripción inversa.- Mg 2+ . In a particular embodiment, the common buffer for both reactions comprises 10 mM Tris-HCl, pH 8.3, 50 mM KC1, the MuLV reverse transcriptase, the human placenta ribonuclease inhibitor, an active DNA polymerase at temperatures above 50 ° C and inactive at temperatures between 37 and 50 ° C, MgCl 2 at a concentration between 1.5 and 5 mM, and a pair of oligonucleotide primers for enzymatic amplification by PCR flanking an identifying region of the viral particle or subviral, of which the reverse initiator oligonucleotide also acts as a reverse transcription initiator.
Los reactivos necesarios para las reacciones de transcripción inversa y amplificación enzimática por PCR son productos comerciales o, en el caso de los iniciadores, son oligonucleótidos con unas secuencias concretas que 14 pueden ser sintetizados mediante técnicas convencionales. Estos reactivos pueden añadirse por separado a los tubos del termociclador que contienen las partículas capturadas o bien pueden mezclarse previamente entre sí (todos o algunos de ellos) y formar una mezcla o cóctel de reactivos que se añade a tales tubos.The reagents necessary for the reactions of reverse transcription and enzymatic amplification by PCR are commercial products or, in the case of the initiators, they are oligonucleotides with specific sequences that 14 can be synthesized by conventional techniques. These reagents can be added separately to the thermocycler tubes containing the captured particles or they can be pre-mixed with each other (all or some of them) and form a mixture or cocktail of reagents that is added to such tubes.
C) Ciclo térmico La tercera etapa comprende introducir los tubos del termociclador que contienen las partículas virales o subvirales capturadas junto con los reactivos necesarios para las reacciones de transcripción inversa y de amplificación enzimática por PCR, y aplicar a dichos tubos un ciclo térmico adecuado para realizar, en primer lugar, la reacción de transcripción inversa, y finalizada ésta, la reacción de amplificación enzimática por PCR del fragmento de ácido nucleico seleccionado, sin retirar el tubo del termociclador, sin abrir los tubos que contienen las muestras del ensayo y sin más que modificar la temperatura. Para ello, el termociclador se programa para que realice un ciclo térmico que comprende: calentar los tubos del termociclador a una temperatura y durante un periodo de tiempo adecuados para que tenga lugar la reacción de transcripción inversa, - calentar los tubos a una temperatura y durante un periodo de tiempo adecuados para inactivar la transcriptasa inversa; yC) Thermal cycle The third stage comprises introducing the tubes of the thermal cycler containing the viral or subviral particles captured together with the reagents necessary for the reactions of reverse transcription and enzymatic amplification by PCR, and applying to said tubes a suitable thermal cycle to perform First, the reverse transcription reaction, and after completion of this, the enzymatic PCR amplification reaction of the selected nucleic acid fragment, without removing the tube from the thermal cycler, without opening the tubes containing the test samples and with no more than modify the temperature For this, the thermal cycler is programmed to carry out a thermal cycle comprising: heating the thermocycler tubes at a temperature and for a period of time suitable for the reverse transcription reaction to take place, - heating the tubes at a temperature and during a suitable period of time to inactivate reverse transcriptase; Y
- realizar un número variable y repetido de ciclos de PCR a las temperaturas y tiempos de desnaturalización, anillamiento y elongación adecuados.- perform a variable and repeated number of PCR cycles at the appropriate temperatures and times of denaturation, banding and elongation.
En general, la transcripción inversa se realiza a la temperatura de funcionamiento de la transcriptasa inversa, que, en una realización particular de esta invención será igual o inferior a 50°C, normalmente entre 37 y 50°C, durante un periodo de tiempo comprendido entre 20 y 90 15 minutos .In general, reverse transcription is performed at the operating temperature of the reverse transcriptase, which, in a particular embodiment of this invention will be equal to or less than 50 ° C, usually between 37 and 50 ° C, for a period of time comprised between 20 and 90 15 minutes .
A continuación, los tubos se calientan hasta una temperatura adecuada para desnaturalizar térmicamente la transcriptasa inversa y, en caso de utilizar una DNA polimerasa termoestable activable a temperatura elevada, activar dicha polimerasa. En una realización particular, los tubos se calientan a una temperatura superior a 90°C, durante un periodo de tiempo comprendido entre 2 y 10 minutos, con lo que se inactiva térmicamente la transcriptasa inversa, se activa la DNA polimerasa (en su caso) y se desnaturaliza el híbrido RNA-DNA formado para que pueda comenzar la reacción de amplificación enzimática por PCR.The tubes are then heated to a temperature suitable for thermally denaturing the reverse transcriptase and, if using a thermostable DNA polymerase activatable at elevated temperature, activating said polymerase. In a particular embodiment, the tubes are heated to a temperature greater than 90 ° C, for a period of time between 2 and 10 minutes, whereby the reverse transcriptase is thermally inactivated, the DNA polymerase is activated (if any) and the RNA-DNA hybrid formed is denatured so that the PCR amplification reaction can begin.
La reacción de amplificación enzimática por PCR implica la realización de un número adecuado de ciclos repetidos de calentamiento y enfriamiento, comprendiendo cada ciclo las etapas de:The enzymatic PCR amplification reaction involves performing an adequate number of repeated heating and cooling cycles, each cycle comprising the steps of:
- desnaturalización térmica del DNA bicatenario, que depende de la temperatura de separación de las cadenas del DNA bicatenario formado;- thermal denaturation of double stranded DNA, which depends on the separation temperature of the strands of the double stranded DNA formed;
- anillamiento de los oligonucleótidos iniciadores que flanquean el fragmento de DNA a amplificar, que depende de la temperatura de hibridación de dichos iniciadores al DNA molde; y - elongación de los iniciadores anillados con la DNA polimerasa, que depende de la temperatura de funcionamiento de la DNA polimerasa termoestable.- banding of the initiating oligonucleotides flanking the DNA fragment to be amplified, which depends on the hybridization temperature of said initiators to the template DNA; and - elongation of the initiators ringed with DNA polymerase, which depends on the operating temperature of the thermostable DNA polymerase.
En ocasiones, cuando la temperatura de anillamiento es igual o superior a 60°C se pueden realizar simultáneamente las etapas de anillamiento y elongación.Sometimes, when the banding temperature is equal to or greater than 60 ° C, the banding and elongation stages can be performed simultaneously.
El número de ciclos a realizar depende del grado de amplificación del fragmento de ácido nucleico que se pretende obtener. En general, se obtienen unos resultados adecuados con un número de ciclos comprendido entre 25 y 50. 16The number of cycles to be performed depends on the degree of amplification of the nucleic acid fragment that is intended to be obtained. In general, adequate results are obtained with a number of cycles between 25 and 50. 16
Finalmente, la mezcla de reacción se mantiene a la temperatura de elongación durante un periodo de tiempo prolongado para terminar la reacción de amplificación enzimática por PCR. En una realización particular de esta invención, dicho ciclo térmico comprende: a) calentar la mezcla de reacción, formada por las partículas virales o subvirales capturadas junto con los reactivos para las reacciones de transcripción inversa y amplificación enzimática por PCR, a una temperatura comprendida entre 37 y 48°C, durante 30 minutos, con el fin de efectuar la reacción de transcripción inversa, a continuación, b) calentar la mezcla de reacción a una temperatura comprendida entre 92 y 97°C, durante 5 minutos, para inactivar la transcriptasa inversa, desnaturalizar el híbrido RNA-DNA y, en su caso, activar la DNA polimerasa termoestable; c) repetir un número variable de veces, normalmente entre 25 y 50 veces, un ciclo de calentamiento y enfriamiento, que comprende las etapas de:Finally, the reaction mixture is maintained at the elongation temperature for a prolonged period of time to terminate the PCR amplification reaction. In a particular embodiment of this invention, said thermal cycle comprises: a) heating the reaction mixture, formed by the viral or subviral particles captured together with the reagents for the reactions of reverse transcription and enzymatic amplification by PCR, at a temperature between 37 and 48 ° C, for 30 minutes, in order to effect the reverse transcription reaction, then b) heat the reaction mixture at a temperature between 92 and 97 ° C, for 5 minutes, to inactivate the transcriptase conversely, denature the RNA-DNA hybrid and, where appropriate, activate the thermostable DNA polymerase; c) repeat a variable number of times, usually between 25 and 50 times, a heating and cooling cycle, comprising the steps of:
- desnaturalización: normalmente a una temperatura superior a 90°C, en general comprendida entre 92 y 97°C, durante un periodo de tiempo superior a 15 segundos; - anillamiento: a la temperatura de hibridación de los iniciadores al DNA molde; y- denaturation: normally at a temperature greater than 90 ° C, generally between 92 and 97 ° C, for a period of time greater than 15 seconds; - banding: at the hybridization temperature of the initiators to the template DNA; Y
- elongación: a una temperatura comprendida entre aproximadamente 60 y 94 °C, durante un periodo de tiempo normalmente superior a 45 segundos; y d) al terminar el último ciclo, mantener la temperatura de elongación durante un periodo de tiempo normalmente superior a 2 minutos con el fin de terminar la reacción de amplificación enzimática por PCR.- elongation: at a temperature between approximately 60 and 94 ° C, for a period of time normally greater than 45 seconds; and d) at the end of the last cycle, maintain the elongation temperature for a period of time normally exceeding 2 minutes in order to terminate the PCR amplification reaction.
Los fragmentos así amplificados pueden ser sometidos a un análisis por técnicas espectrofotométricas y/o 17 electroforéticas con el fin de detectar, cuantificar y/o identificar la partícula viral o subviral en cuestión, o alternativamente pueden ser sometidos a un análisis estructural bien por técnicas de secuenciación de ácidos nucleicos (Maxam, A. M., y Gilbert, ., (1977), Proc. Nati. Acad. Sci. USA, 74:560; y Sanger, F. , et al., (1977), Proc. Nati. Acad. Sci. USA, 74:5463) o por análisis de polimorfismos de longitud de fragmentos de restricción (RFLP) (Tanskley, S. D., et al., (1989), Biotecnology, 7:253; Gillings, M., et al., (1993), J. Virol. Methods,The fragments thus amplified can be subjected to analysis by spectrophotometric and / or techniques. 17 electrophoretic in order to detect, quantify and / or identify the viral or subviral particle in question, or alternatively they can be subjected to a structural analysis either by nucleic acid sequencing techniques (Maxam, AM, and Gilbert,., (1977 ), Proc. Nati. Acad. Sci. USA, 74: 560; and Sanger, F., et al., (1977), Proc. Nati. Acad. Sci. USA, 74: 5463) or by analysis of polymorphisms of restriction fragment length (RFLP) (Tanskley, SD, et al., (1989), Biotechnology, 7: 253; Gillings, M., et al., (1993), J. Virol. Methods,
44:305; Blanco Urgoiti et al., (1996), Archives of44: 305; Blanco Urgoiti et al., (1996), Archives of
Virology, en prensa) o de conformación de cadena sencillaVirology, in press) or single chain conformation
(SSCP) (Orita, M. , et al., (1989), Genomics, 5, 874-879), con el fin de tipificar molecularmente la partícula viral o subviral y realizar estudios de epidemiología molecular. Los siguientes ejemplos sirven para ilustrar la invención.(SSCP) (Orita, M., et al., (1989), Genomics, 5, 874-879), in order to molecularly typify the viral or subviral particle and conduct molecular epidemiology studies. The following examples serve to illustrate the invention.
EJEMPLO 1 Detección e identificación del virus Y (PVY) de la patataEXAMPLE 1 Detection and identification of potato virus Y (PVY)
En este ejemplo se describe la aplicación del procedimiento de esta invención para llevar a cabo la detección e identificación del virus Y de la patata (PVY) .This example describes the application of the method of this invention to carry out the detection and identification of the potato virus Y (PVY).
Muestras de hojas de patata procedentes de plantas sanas y de plantas infectadas con PVY se homogenizan, por separado, en una relación 1/10 (peso/volumen) con un tampón de extracción compuesto por Tris-HCl 0,5 M, pH 8,0, conteniendo 2% de polivinil-pirrolidona, 1% de polietilenglicol 6000, 0,8% de NaCl, 0,005% de T EEN-20® (SIGMA CHEMICAL CO., EEUU) y 0,02% de azida sódica.Samples of potato leaves from healthy plants and plants infected with PVY are homogenized, separately, in a 1/10 ratio (weight / volume) with an extraction buffer composed of 0.5 M Tris-HCl, pH 8, 0, containing 2% polyvinyl pyrrolidone, 1% polyethylene glycol 6000, 0.8% NaCl, 0.005% T EEN-20® (SIGMA CHEMICAL CO., USA) and 0.02% sodium azide.
Alícuotas de 100 microlitros (μl) , se añaden sobre cada tubo de un termociclador GeneAmp® PCR System 9600 (PERKIN-ELMER CETUS) , previamente tapizados con el anticuerpo monoclonal contra PVY, PVY-10E3 (INGENASA, España) , siguiendo el protocolo recomendado por el 18 fabricante.Aliquots of 100 microliters (μl) are added on each tube of a GeneAmp® PCR System 9600 thermal cycler (PERKIN-ELMER CETUS), previously upholstered with the monoclonal antibody against PVY, PVY-10E3 (INGENASA, Spain), following the recommended protocol for him 18 manufacturer.
A continuación, se añaden:Then they are added:
- 8 μl de tampón para la reacción constituido por Tris-HCl 100 mM, pH 8,3, KC1 500 mM, - 50 unidades de transcriptasa inversa del MuLV (PERKIN-ELMER, EEUU) [1 μl de transcriptasa inversa de MuLV a 50 unidades/μl] ,- 8 μl of buffer for the reaction consisting of 100 mM Tris-HCl, pH 8.3, 500 mM KC1, - 50 units of MuLV reverse transcriptase (PERKIN-ELMER, USA) [1 μl of MuLV reverse transcriptase at 50 units / μl],
- 20 unidades de HPRI (AMERSHAM, EEUU), [1 μl de HPRI a 200 unidades/μl] , - 2,5 unidades de AmpliTaq Gold® (PERKIN-ELMER CETUS, EEUU), [0,5 μl de de AmpliTaq Gold® a 50 unidades/μl],- 20 units of HPRI (AMERSHAM, USA), [1 μl of HPRI at 200 units / μl], - 2.5 units of AmpliTaq Gold® (PERKIN-ELMER CETUS, USA), [0.5 μl of AmpliTaq Gold ® at 50 units / μl],
- MgCl2, [entre 6,4 μl y 9,6 μl de MgCl2 25 mM] ,- MgCl 2 , [between 6.4 μl and 9.6 μl of 25 mM MgCl 2 ],
- dNTPs (dATP, dCTP, dGTP y dTTP) , [2 μl de mezcla de dNTPs a 10 mM de cada uno de ellos] (equivale a 0,25 mM) , - iniciador directo (5') 2 μl a 10 μM (0,25 mM) ,- dNTPs (dATP, dCTP, dGTP and dTTP), [2 μl of mixture of dNTPs at 10 mM of each one] (equivalent to 0.25 mM), - direct initiator (5 ') 2 μl to 10 μM ( 0.25 mM),
- iniciador inverso (3') 2 μl a 10 μM (0,25 mM) , y- reverse initiator (3 ') 2 μl to 10 μM (0.25 mM), and
- agua, hasta completar el volumen de 80 μl.- water, until the volume of 80 μl is completed.
Los iniciadores utilizados se recogen en el apartado relativo a la LISTA DE SECUENCIAS, en donde el iniciador directo (5') corresponde al identificado como SEC. ID. N° : 1 y el inverso (3') al identificado como SEC. ID. N°: 2.The initiators used are listed in the section on the LIST OF SEQUENCES, where the direct initiator (5 ') corresponds to the one identified as SEC. ID. N °: 1 and the inverse (3 ') to that identified as SEC. ID. N °: 2.
Estos iniciadores abarcan un fragmento de 879 nucleótidosThese primers encompass an 879 nucleotide fragment
(nt) que corresponden al gen de la proteína de la cápsida más el principio de la región 3' no traducible (3'-utr). El iniciador directo se extiende desde el nt 3869 hasta el nt 3891 del genoma del PVY, mientras que el iniciador inverso se extiende desde el nt 4745 hasta el nt 4723. La secuencia del genoma del PVY ha sido descrita por Hidaka et al., [Nucleic Acid Research, 20, 3515, (1992)]. Para la realización de este Ejemplo se ensayaron 5 concentraciones distintas de MgCl2 (2, 2,25, 2,5, 2,75 y 3 mM) con fines comparativos.(nt) corresponding to the capsid protein gene plus the principle of the 3 'non-translatable region (3'-utr). The direct initiator extends from nt 3869 to nt 3891 of the PVY genome, while the reverse initiator extends from nt 4745 to nt 4723. The PVY genome sequence has been described by Hidaka et al., [ Nucleic Acid Research, 20, 3515, (1992)]. For the realization of this Example, 5 different concentrations of MgCl 2 (2, 2.25, 2.5, 2.75 and 3 mM) were tested for comparative purposes.
A continuación, se introducen los tubos en el termociclador donde se someten al siguiente ciclo térmico: a) calentamiento a 42°C, durante 30 minutos, con el 19 fin de realizar la reacción de transcripción inversa, b) calentamiento a 95°C, durante 5 minutos, para inactivar la transcriptasa inversa y el inhibidor de ribonucleasas, activar la DNA polimerasa, y desnaturalizar el híbrido RNA-DNA, c) 35 ciclos de PCR comprendiendo las etapas de:The tubes are then introduced into the thermal cycler where they undergo the following thermal cycle: a) heating at 42 ° C, for 30 minutes, with the 19 to carry out the reverse transcription reaction, b) heating at 95 ° C, for 5 minutes, to inactivate the reverse transcriptase and the ribonuclease inhibitor, activate the DNA polymerase, and denature the RNA-DNA hybrid, c) 35 cycles PCR comprising the steps of:
- desnaturalización: 45 segundos a 94°C;- denaturation: 45 seconds at 94 ° C;
- anillamiento: 1 minuto a 53°C; y- banding: 1 minute at 53 ° C; Y
- elongación: 1 minuto a 72°C, y d) finalmente, mantenimiento de la reacción a 72°C durante 5 minutos para terminar la amplificación enzimática por PCR.- elongation: 1 minute at 72 ° C, and d) finally, maintaining the reaction at 72 ° C for 5 minutes to complete the enzymatic amplification by PCR.
Las muestras amplificadas se sometieron a una electroforesis en gel de agarosa al 0,8% en tampón TAE como indica Sambrook et al., (1989) [Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor] , se tiñeron con una solución de bromuro de etidio (4 μg/μl) y se fotografiaron con una cámara Polaroid sobre un transiluminador de luz ultravioleta con un filtro naranja delante del objetivo.The amplified samples were subjected to 0.8% agarose gel electrophoresis in TAE buffer as indicated by Sambrook et al., (1989) [Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor ], were stained with a solution of ethidium bromide (4 μg / μl) and photographed with a Polaroid camera on an ultraviolet light transilluminator with an orange filter in front of the lens.
Los resultados de la electroforesis se muestran en la Figura 1, donde se observa la presencia de una banda de 879 nt correspondientes al fragmento amplificado del genoma del PVY en las 5 muestras con planta infectada mientras que en las pruebas realizadas con muestras de planta sana no aparece dicha banda.The results of the electrophoresis are shown in Figure 1, where the presence of a band of 879 nt corresponding to the amplified fragment of the PVY genome is observed in the 5 samples with infected plant while in tests performed with samples of healthy plant not this band appears.
EJEMPLO 2 (Comparativo)EXAMPLE 2 (Comparative)
En este Ejemplo se compara la eficacia del método proporcionado por esta invención con el método descrito en la patente española ES 9201232.In this Example, the effectiveness of the method provided by this invention is compared with the method described in Spanish patent ES 9201232.
2.1 Detección e identificación del PVY según el método proporcionado por esta invención, utilizando la polimerasa Taq 202.1 Detection and identification of PVY according to the method provided by this invention, using Taq polymerase twenty
Muestras de hojas de patata procedentes de plantas sanas y de plantas infectadas con PVY se homogenizan según la metodología descrita en el Ejemplo 1. Posteriormente, se añaden alícuotas de 100 μl sobre los tubos de un termociclador GeneAmp® PCR System 9600 (PERKIN-ELMER CETUS) , previamente tapizados con el anticuerpo monoclonal contra PVY, PVY-10E3 (INGENASA, España), siguiendo el protocolo recomendado por el fabricante. A continuación, se añaden: - 8 μl de un tampón constituido por Tris-HCl 100 mM, pH 8,3, KC1 500 mM,Samples of potato leaves from healthy plants and plants infected with PVY are homogenized according to the methodology described in Example 1. Subsequently, 100 μl aliquots are added on the tubes of a GeneAmp® PCR System 9600 thermal cycler (PERKIN-ELMER CETUS ), previously upholstered with the monoclonal antibody against PVY, PVY-10E3 (INGENASA, Spain), following the protocol recommended by the manufacturer. The following are added: - 8 μl of a buffer consisting of 100 mM Tris-HCl, pH 8.3, 500 mM KC1,
50 unidades de transcriptasa inversa del MuLV (PERKIN-ELMER, EEUU) [1 μl de transcriptasa inversa de MuLV a 50 unidades/μl], - 20 unidades de HPRI (AMERSHAM, EEUU), [1 μl de HPRI a 200 unidades/μl] ,50 units of MuLV reverse transcriptase (PERKIN-ELMER, USA) [1 μl of MuLV reverse transcriptase at 50 units / μl], - 20 units of HPRI (AMERSHAM, USA), [1 μl of HPRI at 200 units / μl ],
- 2,5 unidades de AmpliTaq (PERKIN-ELMER CETUS, EEUU), [0,5 μl de de AmpliTaq Gold® a 50 unidades/μl],- 2.5 units of AmpliTaq (PERKIN-ELMER CETUS, USA), [0.5 μl of AmpliTaq Gold® at 50 units / μl],
- MgCl2, - dNTPs (dATP, dCTP, dGTP y dTTP) , [2 μl de mezcla de dNTPs a 10 mM de cada uno de ellos] (equivale a 0,25 mM) ,- MgCl 2 , - dNTPs (dATP, dCTP, dGTP and dTTP), [2 μl of mixture of dNTPs at 10 mM of each one] (equivalent to 0.25 mM),
- iniciador directo (5') 2 μl a 10 μM (0,25 mM) ,- direct initiator (5 ') 2 μl to 10 μM (0.25 mM),
- iniciador inverso (3') 2 μl a 10 μM (0,25 mM) , y- reverse initiator (3 ') 2 μl to 10 μM (0.25 mM), and
- agua, hasta completar un volumen ,de 80 μl . Los iniciadores utilizados son los mismos que los del Ejemplo 1.- water, until a volume of 80 μl is completed. The primers used are the same as those in Example 1.
En este Ejemplo se ensayaron 2 concentraciones distintas de MgCl2 (3 y 5 mM) con fines comparativos.In this Example, 2 different concentrations of MgCl 2 (3 and 5 mM) were tested for comparative purposes.
Posteriormente, los tubos se introducen en el termo- ciclador donde se someten al siguiente ciclo térmico: a) calentamiento a 42°C, durante 30 minutos, con el fin de realizar la reacción de transcripción inversa, b) calentamiento a 97°C, durante 5 minutos, para inactivar la transcriptasa inversa y el inhibidor de ribonucleasas, y desnaturalizar el híbrido RNA-DNA, 21 c) 35 ciclos de PCR comprendiendo las etapas de:Subsequently, the tubes are introduced into the thermal cycler where they undergo the following thermal cycle: a) heating at 42 ° C, for 30 minutes, in order to carry out the reverse transcription reaction, b) heating at 97 ° C, for 5 minutes, to inactivate the reverse transcriptase and the ribonuclease inhibitor, and denature the RNA-DNA hybrid, 21 c) 35 cycles of PCR comprising the steps of:
- desnaturalización: 20 segundos a 94°C;- denaturation: 20 seconds at 94 ° C;
- anillamiento: 20 segundos a 53°C; y- banding: 20 seconds at 53 ° C; Y
- elongación: 1 minuto a 72°C, y d) finalmente, mantenimiento de la reacción a 72°C durante 5 minutos para terminar la amplificación enzimática por PCR.- elongation: 1 minute at 72 ° C, and d) finally, maintaining the reaction at 72 ° C for 5 minutes to complete the enzymatic amplification by PCR.
2.2 Detección e identificación del PVY mediante el método de la patente española ES 9201232 utilizando la polimerasa Taq2.2 Detection and identification of PVY by the method of Spanish patent ES 9201232 using Taq polymerase
Muestras de hojas de patata procedentes de plantas sanas y de plantas infectadas con PVY se homogenizan según la metodología descrita en el Ejemplo 1. A continuación, se añaden alícuotas de 100 μl sobre los tubos de un termociclador GeneAmp® PCR System 9600 (PERKIN-ELMER CETUS), previamente tapizados con el anticuerpo monoclonal contra PVY, PVY-10E3 (INGENASA, España), siguiendo el protocolo recomendado por el fabricante. 2.2.1 Transcripción inversaSamples of potato leaves from healthy plants and plants infected with PVY are homogenized according to the methodology described in Example 1. Next, 100 μl aliquots are added on the tubes of a GeneAmp® PCR System 9600 thermal cycler (PERKIN-ELMER CETUS), previously upholstered with the monoclonal antibody against PVY, PVY-10E3 (INGENASA, Spain), following the protocol recommended by the manufacturer. 2.2.1 Reverse transcription
La reacción de transcripción inversa se lleva a cabo en un volumen final de 20 μl que contenía:The reverse transcription reaction is carried out in a final volume of 20 μl containing:
- un tampón constituido por Tris-HCl 10 mM, pH 8,3, KC1 50 mM, - MgCl2 5 mM,- a buffer consisting of 10 mM Tris-HCl, pH 8.3, 50 mM KC1, - 5 mM MgCl 2 ,
- iniciador inverso (3') 0,25 μM,- reverse initiator (3 ') 0.25 μM,
- dNTPs 0,25 mM,- 0.25 mM dNTPs,
- 20 unidades de HPRI (AMERSHAM, EEUU) ,- 20 units of HPRI (AMERSHAM, USA),
50 unidades de transcriptasa inversa del MuLV (PERKIN-ELMER, EEUU) y50 units of MuLV reverse transcriptase (PERKIN-ELMER, USA) and
- agua.- Water.
El iniciador inverso (3') utilizado es el mismo que el del Ejemplo 1.The reverse initiator (3 ') used is the same as in Example 1.
La reacción de transcripción inversa se llevó a cabo calentando a 42°C durante 30 minutos y posteriormente los 22 tubos se colocaron en hielo.The reverse transcription reaction was carried out by heating at 42 ° C for 30 minutes and subsequently 22 tubes were placed on ice.
2.2.2 PCR Para efectuar la PCR es necesario añadir a los tubos los productos que se indican a continuación. La PCR se realizó con un volumen final de 80 μl para lo cual se añade:2.2.2 PCR To carry out the PCR it is necessary to add the products indicated below to the tubes. The PCR was performed with a final volume of 80 μl to which is added:
- un tampón constituido por Tris-HCl 10 mM, pH 8,3, KC1 50 mM,- a buffer consisting of 10 mM Tris-HCl, pH 8.3, 50 mM KC1,
- iniciador directo (5') 0,25 μM, - 2,5 unidades de A plitaq® (PERKIN-ELMER, EEUU)- direct initiator (5 ') 0.25 μM, - 2.5 units of A plitaq® (PERKIN-ELMER, USA)
- MgCl2 (hasta alcanzar una concentración de 3 mM, teniendo en cuenta el Mg2+ añadido para la transcripción inversa y que ahora se lleva hasta un volumen de 80 μl) .- MgCl 2 (until reaching a concentration of 3 mM, taking into account the Mg 2+ added for reverse transcription and which is now brought to a volume of 80 μl).
El iniciador directo (5') utilizado es el mismo que el del Ejemplo 1.The direct initiator (5 ') used is the same as in Example 1.
Posteriormente, los tubos se introducen en el termociclador donde se someten al siguiente ciclo térmico: a) calentamiento a 97°C, durante 5 minutos, b) 35 ciclos de PCR comprendiendo las etapas de: - desnaturalización: 20 segundos a 94°C;Subsequently, the tubes are introduced into the thermal cycler where they undergo the following thermal cycle: a) heating at 97 ° C, for 5 minutes, b) 35 PCR cycles comprising the steps of: - denaturation: 20 seconds at 94 ° C;
- anillamiento: 20 segundos a 53°C; y- banding: 20 seconds at 53 ° C; Y
- elongación: 1 minuto a 72°C, y c) finalmente, mantenimiento de la reacción a 72°C durante 5 minutos para terminar la amplificación enzimática por PCR.- elongation: 1 minute at 72 ° C, and c) finally, maintaining the reaction at 72 ° C for 5 minutes to complete the enzymatic amplification by PCR.
Las productos resultantes de la reacción de amplificación procedentes tanto del Ejemplo 2.1 como del Ejemplo 2.2 se sometieron a electroforesis en gel de agarosa tal como se describe en el Ejemplo 1. Los resultados obtenidos se muestran en la Figura 2, donde se observa que el rendimiento obtenido con la reacción de transcripción inversa-PCR en un solo paso a una concentración 3 mM de MgCl2 (Ejemplo 2.1: método proporcionado por la invención) es varias veces superior al obtenido según el procedimiento descrito en la patente 23The products resulting from the amplification reaction from both Example 2.1 and Example 2.2 were subjected to agarose gel electrophoresis as described in Example 1. The results obtained are shown in Figure 2, where it is observed that the yield obtained with the one-step reverse transcription-PCR reaction at a 3 mM concentration of MgCl 2 (Example 2.1: method provided by the invention) is several times higher than that obtained according to the procedure described in the patent 2. 3
española ES 9201232 (Ejemplo 2.2). La mayor brillantez de la banda visible en el carril correspondiente al producto amplificado según el Ejemplo 2.1 con una concentración de MgCl2 de 3 mM es indicativa de la mayor cantidad de producto amplificado.Spanish ES 9201232 (Example 2.2). The greater brightness of the visible band in the lane corresponding to the amplified product according to Example 2.1 with a MgCl 2 concentration of 3 mM is indicative of the greater quantity of amplified product.
HOJA DE SUSTITUCIÓN (REGLA 26) 24SUBSTITUTE SHEET (RULE 26) 24
LISTA DE SECUENCIASLIST OF SEQUENCES
(1) INFORMACIÓN GENERAL:(1. GENERAL INFORMATION:
(i) SOLICITANTE:(i) APPLICANT:
(A) NOMBRE: INSTITUTO NACIONAL DE INVESTIGACIÓN Y TECNOLOGÍA AGRARIA Y ALIMENTARIA (INIA) (B) CALLE: José Abascal, 56(A) NAME: NATIONAL INSTITUTE OF RESEARCH AND AGRICULTURAL AND FOOD TECHNOLOGY (INIA) (B) STREET: José Abascal, 56
(C) CIUDAD: Madrid(C) CITY: Madrid
(D) ESTADO: Madrid(D) STATE: Madrid
(E) PAÍS: ES(E) COUNTRY: EN
(F) CÓDIGO POSTAL (ZIP) : 28003 (H) TELEFONO: 91 347 39 33(F) ZIP CODE: 28003 (H) PHONE: 91 347 39 33
(I) TELEFAX: 91 347 39 31(I) TELEFAX: 91 347 39 31
(ii) TITULO DE LA INVENCIÓN: PROCEDIMIENTO PARA LA OBTENCIÓN DE FRAGMENTOS AMPLIFICADOS DE ACIDO NUCLEICO DE PARTÍCULAS VIRALES Y SUBVIRALES QUE CONTIENEN RNA(ii) TITLE OF THE INVENTION: PROCEDURE FOR OBTAINING AMPLIFIED FRAGMENTS OF NUCLEIC ACID OF VIRAL AND SUBVIRAL PARTICLES CONTAINING RNA
(iii) NUMERO DE SECUENCIAS: 2(iii) NUMBER OF SEQUENCES: 2
(iv) FORMA LEGIBLE POR ORDENADOR:(iv) LEGIBLE FORM BY COMPUTER:
(A) TIPO DE SOPORTE: Floppy disk(A) SUPPORT TYPE: Floppy disk
(B) ORDENADOR: IBM PC compatible (C) SISTEMA OPERATIVO: PC-DOS/MS-DOS(B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS
(D) SOFTWARE: Patentln Reléase #1.0, Versión #1.30 (OEP)(D) SOFTWARE: Patentln Relay # 1.0, Version # 1.30 (EPO)
HOJA DE SUSTITUCIÓN (REGLA 26) 24 bisSUBSTITUTE SHEET (RULE 26) 24 bis
(2) INFORMACIÓN DE LA SECUENCIA IDENTIFICADA N° 1 (SEC. ID. N° 1) :(2) IDENTIFIED SEQUENCE INFORMATION N ° 1 (SEQ. ID. N ° 1):
(i) CARACTERÍSTICAS DE LA SECUENCIA: (A) LONGITUD: 23 pares de bases(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 base pairs
(B) TIPO: ácido nucleico(B) TYPE: nucleic acid
(C) NUMERO DE CADENAS: sencilla(C) NUMBER OF CHAINS: simple
(D) TOPOLOGÍA: lineal(D) TOPOLOGY: linear
(ii) TIPO DE MOLÉCULA: ADN (genómico)(ii) TYPE OF MOLECULE: DNA (genomic)
(xi) DESCRIPCIÓN DE LA SECUENCIA: SEC. ID. N° 1(xi) SEQUENCE DESCRIPTION: SEC. ID. No. 1
TAAAAGTAGT ACAGGAAAAG CCA 23TAAAAGTAGT ACAGGAAAAG CCA 23
(2) INFORMACIÓN DE LA SECUENCIA IDENTIFICADA N° 2 (SEC. ID. N° 2) :(2) IDENTIFIED SEQUENCE INFORMATION N ° 2 (SEQ. ID. N ° 2):
(i) CARACTERÍSTICAS DE LA SECUENCIA: (A) LONGITUD: 23 pares de bases(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 base pairs
(B) TIPO: ácido nucleico(B) TYPE: nucleic acid
(C) NUMERO DE CADENAS: sencilla(C) NUMBER OF CHAINS: simple
(D) TOPOLOGÍA: lineal(D) TOPOLOGY: linear
(ii) TIPO DE MOLÉCULA: ADN (genómico)(ii) TYPE OF MOLECULE: DNA (genomic)
(xi) DESCRIPCIÓN DE LA SECUENCIA: SEC. ID. N° 2:(xi) SEQUENCE DESCRIPTION: SEC. ID. N ° 2:
AAATGACACA ATTGATGCAG GAG 23AAATGACACA ATTGATGCAG GAG 23
HOJA DE SUSTITUCIÓN (REGLA SUBSTITUTE SHEET (RULE

Claims

25 REIVINDICACIONES 25 CLAIMS
1. Un procedimiento para la obtención de fragmentos amplificados de ácido nucleico de partículas virales o subvirales que contienen RNA presentes en una muestra sospechosa de contener tales partículas que comprende las etapas de: a) añadir dicha muestra a los tubos de un termociclador e incubar el conjunto bajo condiciones adecuadas para capturar dichas partículas virales o subvirales en los tubos del termociclador; b) retirar el sobrenadante de los tubos y lavar el conjunto de partículas virales o subvirales capturadas en los tubos del termociclador; c) añadir a dichos tubos, que contienen las partículas capturadas, todos los reactivos necesarios para realizar la reacción de transcripción inversa y la reacción de amplificación enzimática por PCR; d) introducir los tubos en el termociclador; y e) programar en el termociclador un ciclo térmico adecuado para que tenga lugar, en cada tubo, en primer lugar, la reacción de transcripción inversa del RNA, y posteriormente, la reacción de amplificación enzimática por PCR del fragmento de ácido nucleico tras desactivación térmica de la transcriptasa inversa, para lo cual dicho ciclo térmico comprende: calentar los tubos del termociclador a una temperatura y durante un periodo de tiempo adecuados para que tenga lugar la reacción de transcripción inversa, - calentar los tubos a una temperatura y durante un periodo de tiempo adecuados para inactivar la transcriptasa inversa; y1. A method for obtaining amplified nucleic acid fragments of viral or subviral particles containing RNA present in a sample suspected of containing such particles comprising the steps of: a) adding said sample to the tubes of a thermal cycler and incubating the set under suitable conditions to capture said viral or subviral particles in the thermocycler tubes; b) remove the supernatant from the tubes and wash the set of viral or sub-viral particles captured in the tubes of the thermal cycler; c) adding to said tubes, which contain the captured particles, all the reagents necessary to carry out the reverse transcription reaction and the enzymatic amplification reaction by PCR; d) insert the tubes into the thermal cycler; and e) program a suitable thermal cycle in the thermal cycler so that the reverse transcription reaction of the RNA takes place in each tube first, and subsequently the enzymatic PCR amplification reaction of the nucleic acid fragment after thermal deactivation of the reverse transcriptase, for which said thermal cycle comprises: heating the thermocycler tubes at a temperature and for a period of time suitable for the reverse transcription reaction to take place, - heating the tubes at a temperature and for a period of time suitable for inactivating reverse transcriptase; Y
- realizar un número variable y repetido de ciclos de PCR a las temperaturas y tiempos de desnaturalización, anillamiento y elongación adecuados. 26- perform a variable and repeated number of PCR cycles at the appropriate temperatures and times of denaturation, banding and elongation. 26
2. Un procedimiento según la reivindicación 1, en el que la inmovilización sobre el tubo del termociclador de las estructuras macromoleculares relacionadas con dichas partículas virales o subvirales se lleva a cabo mediante el empleo de moléculas retenidas sobre dicho tubo que tienen afinidad por tales estructuras macromoleculares.2. A method according to claim 1, wherein immobilization on the thermocycler tube of macromolecular structures related to said viral or subviral particles is carried out by using molecules retained on said tube having affinity for such macromolecular structures .
3. Un procedimiento según la reivindicación 2, en el que dichas moléculas son anticuerpos.3. A method according to claim 2, wherein said molecules are antibodies.
4. Un procedimiento según la reivindicación 3, en el que dichos anticuerpos se seleccionan del grupo formado por:4. A method according to claim 3, wherein said antibodies are selected from the group consisting of:
- anticuerpos frente a proteínas de la cubierta de la partícula viral,- antibodies against proteins of the viral particle envelope,
- anticuerpos frente a proteínas de la cubierta de una célula hospedadora que contiene la partícula viral, y- antibodies against coat proteins of a host cell containing the viral particle, and
- anticuerpos frente a RNA de doble cadena cuando la partícula es una partícula viral o subviral.- antibodies against double stranded RNA when the particle is a viral or subviral particle.
5. Un procedimiento según la reivindicación 1, en el que dichos reactivos necesarios para la realización de las etapas de transcripción inversa y amplificación enzimática por PCR comprenden: - un tampón adecuado para la reacción de transcripción inversa y para la reacción de amplificación enzimática por PCR,5. A method according to claim 1, wherein said reagents necessary for performing the steps of reverse transcription and enzymatic amplification by PCR comprise: - a buffer suitable for the reverse transcription reaction and for the enzymatic amplification reaction by PCR ,
- una transcriptasa inversa,- a reverse transcriptase,
- una DNA polimerasa termoestable, - un inhibidor de ribonucleasas,- a thermostable DNA polymerase, - a ribonuclease inhibitor,
- un oligonucleótido que actúa como iniciador para la reacción de transcripción inversa y como iniciador inverso (3') de la reacción de amplificación enzimática por PCR,- an oligonucleotide that acts as an initiator for the reverse transcription reaction and as a reverse initiator (3 ') of the enzymatic amplification reaction by PCR,
- un oligonucleótido que actúa como iniciador directo (5') para la reacción de amplificación enzimática por PCR; 27- an oligonucleotide that acts as a direct initiator (5 ') for the enzymatic amplification reaction by PCR; 27
- los desoxinucleósidos trifosfato (dATP, dCTP, dGTP y dTTP) ; y- deoxynucleosides triphosphate (dATP, dCTP, dGTP and dTTP); Y
- Mg2+.- Mg 2+ .
6. Un procedimiento según la reivindicación 5, en el que dicho tampón tiene un pH de 8,3.6. A method according to claim 5, wherein said buffer has a pH of 8.3.
7. Un procedimiento según la reivindicación 5, en el que dicha transcriptasa inversa es activa a la temperatura en la que se realiza la reacción de transcripción inversa, pero no es activa a la temperatura en la que se realiza la reacción de amplificación enzimática por PCR.7. A method according to claim 5, wherein said reverse transcriptase is active at the temperature at which the reverse transcription reaction is performed, but is not active at the temperature at which the enzymatic PCR amplification reaction is performed. .
8. Un procedimiento según la reivindicación 5, en el que dicha transcriptasa inversa se selecciona del grupo formado por la transcriptasa inversa del virus de la mieloblastosis de ave (AMV) y la transcriptasa inversa del virus de la leucemia murina (MuLV) .A method according to claim 5, wherein said reverse transcriptase is selected from the group consisting of the reverse transcriptase of the bird myeloblastosis virus (AMV) and the reverse transcriptase of the murine leukemia virus (MuLV).
9. Un procedimiento según la reivindicación 5, en el que dicha DNA polimerasa termoestable es activa a la temperatura en la que se realiza la reacción de transcripción inversa y la reacción de amplificación enzimática por PCR.9. A method according to claim 5, wherein said thermostable DNA polymerase is active at the temperature at which the reverse transcription reaction and the enzymatic amplification reaction by PCR is performed.
10. Un procedimiento según la reivindicación 5, en el que dicha DNA polimerasa termoestable es activa a la temperatura en la que se realiza la reacción de amplificación enzimática por PCR y es inactiva a la temperatura en la que se realiza la reacción de transcripción inversa.10. A method according to claim 5, wherein said thermostable DNA polymerase is active at the temperature at which the enzymatic amplification reaction is performed by PCR and is inactive at the temperature at which the reverse transcription reaction is performed.
11. Un procedimiento según la reivindicación 5, en el que dicho inhibidor de ribonucleasas es el inhibidor de ribonucleasas de placenta humana (HPRI) . 2811. A method according to claim 5, wherein said ribonuclease inhibitor is the human placenta ribonuclease inhibitor (HPRI). 28
12. Un procedimiento según la reivindicación 5, en el que los oligonucleótidos que actúan como iniciadores de la reacción de amplificación enzimática por PCR flanquean un fragmento de DNA identificativo de la familia, especie o tipo, de la partícula viral o subviral, de forma que la presencia de dicho fragmento amplificado es indicativo de la existencia de dicha partícula viral o subviral en la muestra analizada.12. A method according to claim 5, wherein the oligonucleotides that act as initiators of the PCR enzyme amplification reaction flank a DNA fragment identifying the family, species or type, of the viral or subviral particle, so that The presence of said amplified fragment is indicative of the existence of said viral or subviral particle in the analyzed sample.
13. Un procedimiento según la reivindicación 5, en el que el Mg2+ está presente en forma de MgCl2, en una concentración comprendida entre 1,5 y 5 mM.13. A process according to claim 5, wherein the Mg 2+ is present in the form of MgCl 2 , in a concentration between 1.5 and 5 mM.
14. Un procedimiento según la reivindicación 1, en el que la reacción de transcripción inversa se realiza a una temperatura igual o inferior a 50°C.14. A method according to claim 1, wherein the reverse transcription reaction is performed at a temperature equal to or less than 50 ° C.
15. Un procedimiento según la reivindicación 1, en el que dicho ciclo térmico comprende calentar la mezcla de reacción, formada por las partículas virales o subvirales capturadas junto con los reactivos para las reacciones de transcripción inversa y amplificación enzimática por PCR, a una temperatura igual o inferior a 50°C, durante el tiempo necesario para que tenga lugar la reacción de transcripción inversa.15. A method according to claim 1, wherein said thermal cycle comprises heating the reaction mixture, formed by the viral or subviral particles captured together with the reagents for the reverse transcription reactions and enzymatic PCR amplification, at an equal temperature or below 50 ° C, for the time necessary for the reverse transcription reaction to take place.
16. Un procedimiento según la reivindicación 1, en el que dicho ciclo térmico comprende calentar la mezcla de reacción, una vez terminada la reacción de transcripción inversa, a una temperatura superior a 90°C, durante el tiempo suficiente para inactivar la transcriptasa inversa.16. A method according to claim 1, wherein said thermal cycle comprises heating the reaction mixture, once the reverse transcription reaction is finished, at a temperature greater than 90 ° C, for sufficient time to inactivate the reverse transcriptase.
17. Un procedimiento según la reivindicación 1, en el que cada ciclo de PCR comprende las etapas de: - desnaturalización, a la temperatura de separación de 29 las cadenas de DNA;17. A method according to claim 1, wherein each PCR cycle comprises the steps of: - denaturation, at the separation temperature of 29 strands of DNA;
- anillamiento, a la temperatura de hibridación de los oligonucleótidos iniciadores al DNA molde; y- banding, at the hybridization temperature of the initiating oligonucleotides to the template DNA; Y
- elongación, a la temperatura de funcionamiento de la DNA polimerasa termoestable.- elongation, at the operating temperature of the thermostable DNA polymerase.
18. Un procedimiento según la reivindicación 1, en el que cada ciclo de PCR comprende las etapas de:18. A method according to claim 1, wherein each PCR cycle comprises the steps of:
- desnaturalización, a la temperatura de separación de las cadenas de DNA; y- denaturation, at the separation temperature of the DNA chains; Y
- anillamiento-elongación cuando el anillamiento se realiza a una temperatura igual o superior a 60°C. - banding-elongation when the banding is performed at a temperature equal to or greater than 60 ° C.
PCT/ES1998/000128 1996-12-02 1998-05-08 Process for obtaining amplified fragments of nucleic acid of viral and sub-viral particles which contain rna WO1999058725A1 (en)

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ES09602551A ES2121698B1 (en) 1996-12-02 1996-12-02 PROCEDURE FOR THE OBTAINING OF AMPLIFIED FRAGMENTS OF NUCLEIC ACID FROM VIRAL AND SUBVIRAL PARTICLES CONTAINING RNA.
AU70446/98A AU7044698A (en) 1998-05-08 1998-05-08 Process for obtaining amplified fragments of nucleic acid of viral and sub-viralparticles which contain rna
PCT/ES1998/000128 WO1999058725A1 (en) 1996-12-02 1998-05-08 Process for obtaining amplified fragments of nucleic acid of viral and sub-viral particles which contain rna

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US5077192A (en) * 1988-10-25 1991-12-31 The General Hospital Corporation Method of detecting antigenic, nucleic acid-containing macromolecular entities
ES2044784A1 (en) * 1992-06-12 1994-01-01 Inia Procedure for the detection and identification of viral and subviral pathogens.
EP0632134A2 (en) * 1993-07-01 1995-01-04 F. Hoffmann-La Roche Ag Reagents and methods for coupled high temperature reverse transcription and polymerase chain reaction

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JPH04141098A (en) * 1990-09-29 1992-05-14 Shimadzu Corp Reagent for detection of rna and detection of rna using the same reagent

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US5077192A (en) * 1988-10-25 1991-12-31 The General Hospital Corporation Method of detecting antigenic, nucleic acid-containing macromolecular entities
EP0402997A2 (en) * 1989-06-15 1990-12-19 Akzo Nobel N.V. Method for determining nucleic acid
WO1991009944A2 (en) * 1989-12-22 1991-07-11 F.Hoffmann-La Roche Ag High temperature reverse transcriptases
ES2044784A1 (en) * 1992-06-12 1994-01-01 Inia Procedure for the detection and identification of viral and subviral pathogens.
EP0632134A2 (en) * 1993-07-01 1995-01-04 F. Hoffmann-La Roche Ag Reagents and methods for coupled high temperature reverse transcription and polymerase chain reaction

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